All relevant data are within the paper.

Introduction {#sec001}
============

How many genes are involved in the normal ontogeny and physiology of the skin and its adnexal structures? How do they interact and how are they controlled? These questions, simple to articulate, have complex answers at best, but are predicated on our knowledge of gene function and more importantly the pleiotropic functions of genes, which can only be elucidated though experimentation and particularly through genetic manipulation. Faced with this challenge, we might initially search major databases such as Mouse Genome Informatics (MGI; <http://www.informatics.jax.org/>), Online Mendelian Inheritance in Man (<https://www.omim.org/>), or Orphanet (<http://www.orpha.net/>) and ask whether naturally-occurring or genetically engineered mutations show skin and skin-associated phenotypes; this can then be the beginning of a detailed investigation of gene expression, function and control. However, we only have experimentally-derived functional information for about half (11,973) of the protein-coding genes in the mouse as assessed by Gene Ontology annotation (MGI statistics; data accessed 19 Feb 2017) of the estimated 24,211 total, and while there are now many electronically inferred annotations for cellular location, process, and function for many more genes, these are not reliable predictors of the biological process in which genes may be involved. Critically, we have some (in many cases sparse) phenotype information from only 3,710 genes in Online Mendelian Inheritance in Man (accessed 19 Feb 2017). This is therefore greatly enhanced by gene/phenotype data from genetically variant mouse strains for around 9,493 mouse genes with human orthologs. The consequence of this is that although the mouse provides a great deal more functional information than the human, much of the genome remains biologically featureless, its "dark matter", and it is only by mutating genes one by one that we can begin to build up a picture of their involvement in processes and structures, and their patterns of functional pleiotropy which are fundamentally unpredictable from sequence alone. An additional problem with mutant mice made in pursuit of hypothesis-driven science is that they are often made on highly variable or undefined genetic backgrounds, often for particular purposes, without systematic or standardized whole animal phenotyping, making it very difficult to compare phenotypes between mutant mice in any uniform way.

To address this problem on a large-scale basis, an international consortium was formed in 2007 to inactivate ("knockout") all protein-coding genes in the mouse genome in embryonic stem cells using multiple gene targeting approaches on a uniform C57BL/6N genetic background \[[@pone.0180682.ref001], [@pone.0180682.ref002]\]. The second phase of this grand plan, the large-scale production and characterization of these mice, is now underway \[[@pone.0180682.ref003], [@pone.0180682.ref004]\]. The International Mouse Phenotyping Consortium was formed in 2011, composed of 17 Institutes across the world in 11 countries with 5 national funding agencies. This includes the NIH-supported contribution to these efforts, the Knockout Mouse Phenotyping Program, which consists of 3 groups; 1) The Jackson Laboratory, The 2) Baylor College of Medicine, The Wellcome Trust Sanger Institute and MRC Harwell Consortium, and 3) the University of California at Davis, The Toronto Center for Phenogenomics, Children's Hospital Oakland Research Institute, and Charles River Laboratories Consortium). These consortia have generated over 5,000 strains of live mice using these embryonic stem cell lines and have recently transitioned to using CRISPR/Cas9-induced null alleles. Knockout lines are subjected to detailed, systematic evaluation (phenotyping). A majority of the phenotyping tests performed as part of these programs focus on physiological characterization as their primary goal \[[@pone.0180682.ref005]\]. While this approach generates an enormous amount of physiological data (clinical pathology, serology/hematology, behavioral data, etc.) \[[@pone.0180682.ref006], [@pone.0180682.ref007]\], histopathology, especially when done by highly trained experts focusing on specific organs, can often make the direct correlation quickly and accurately, as histologic evaluation by pathologists remains the definitive assay in human and veterinary clinical medicine for making most diagnoses \[[@pone.0180682.ref008], [@pone.0180682.ref009]\]. Furthermore, diseases of some organ systems are only truly defined at the gross clinical level followed by histopathology, special stains, and other tissue based tests, such as immunohistochemistry or electron microscopy. The skin, and its adnexa, is one such system.

While abnormalities of the skin and adnexa (eccrine glands, pilosebaceous unit, hair shaft, and nails) are easily seen by just looking at the mouse with the naked eye or with magnification (dissection or scanning electron microscope), detailed histopathology is still necessary to accurately define the abnormality in the skin and some lesions are only apparent microscopically. A primary high-throughput phenotyping strategy concentrates on wide coverage of a large number of parameters in a large number of mice and consequently is designed to pick up relatively clear-cut examples of phenodeviance.

Prior to the initiation of the current international efforts, both Lexicon and Deltagen undertook large-scale systematic mutagenesis and phenotyping projects, of which Lexicon's used systematic histopathology as a routine phenotyping tool (see \[[@pone.0180682.ref010]\] for discussion) and generated histopathological analyses on nearly 5,000 mutant strains. Detailed descriptions of these strains are now in the process of being published in the literature \[[@pone.0180682.ref011], [@pone.0180682.ref012]\].

More recently, the Sanger Mouse Genetics Program reported a preliminary general histopathology workup of 50 randomly selected knockout lines yielding 2 with skin phenotypes, using single samples of dorsal skin from two males and two female mice \[[@pone.0180682.ref013]\]. This was complemented by a "special skin workup" as part of an extended primary phenotyping pipeline \[[@pone.0180682.ref014]\]. The histopathology protocol involved collecting one piece of dorsal skin, fixing, and processing it routinely to create hematoxylin and eosin stained slides. Nearly 800 mutant mouse skin samples were collected from 562 mutant lines in this high-throughput protocol. One and sometimes two slides with two or three serial sections of dorsal skin from one mouse in the study were provided. Mice were approximately 15 weeks of age. Only 23 lines (4.1%) had some sort of skin phenotype while all the others were normal, implying that many of the genes inactivated had no function in the skin; at least not evident from this screening protocol.

The International Mouse Phenotyping Consortium database currently (accessed 17 February 2017) shows that overall 2.22% of 3,199 lines show abnormalities of skin morphology and 5.35% of 2,971, abnormalities of the adnexa. This group reported no abnormalities of skin physiology in its database to date. A search of the Europhenome database, which overlaps with the International Mouse Phenotyping Consortium, shows 12 genes with annotations on skin morphology and physiology (excluding nociception; 27) of 563 genotypes derived from thesemutagenesis efforts (2.1%). The low number of skin phenotypes in these samples contrasts the frequent reporting of skin and adnexal phenotypes both in human Mendelian diseases and in the gathered data of MGI. Detailed examination of the MGI database shows that 1,069 of 9,045 targeted genes with phenotypes are annotated to either skin morphology or phenotypes; approximately 12%. In OMIM, 1,314/3,710 gene entries with phenotypic annotations are annotated to "abnormality of skin, HP:000095" or its children in the Human Phenotype Ontology database (35%) \[[@pone.0180682.ref015]\]. Assuming that we might expect skin and adnexal phenotypes in both humans and mice to result from loss of function of the same gene, the key question is where are the missing phenotypes? Even considering the exceptionally detailed phenotyping carried out in clinical practice, this is a large discrepancy. One possibility is that in the mouse, loss of function genes involved in the development or function of the skin and adnexa might be embryonic or neonatal lethal. However, this may also be an issue of detection; either through skewed phenotyping (i.e. not focused on the skin or not reported) or failure to detect phenotypes. There are projects associated with the US KnockOut Mouse Program that examine embryonic and perinatal lethality, yet none of them look beyond gross skin phenotype as reasons for early mortality \[[@pone.0180682.ref004]\]. From the reasoning above, we believe this problem can be addressed through careful targeted skin phenotyping.

The Jackson Laboratory is one of three sites in the United States generating knockout mutant mice. Histopathology is an optional parameter in the phenotyping pipeline, and thus is not performed and/or reported universally from individual centers. To better address finding skin lesions, representative retired breeder mice were received, histology was done on skin and nails from multiple sites, and slides were screened by a veterinary pathologist specializing in phenotyping skin mutants. Candidate models were subsequently reviewed by a physician pathologist specializing in dermatopathology. In so doing 169 novel mutant lines were reviewed and of those only one was found to have a relatively minor sebaceous gland abnormality.

Results {#sec002}
=======

Skin, hair follicles, and nail units of both sexes were screened for lesions in 169 novel mutant lines of knockout mice generated at JAX ([Table 1](#pone.0180682.t001){ref-type="table"}). Of these 116 (68.6%) were homozygous and 61 (36.1%) were heterozygous mutant mice. Four lines had both heterozygous and homozygous mice screened (2.4%). Of the heterozygous mice, 62.3% were pre-wean lethal and 11.5% were sub-viable, which is defined as yielding fewer than half of the expected number of homozygotes from an intercross (\<12.5%). For these strains no homozygous mutant mice were evaluated. A total of 743 (2.2 average per line and sex) mice were necropsied and the skin and adnexa were evaluated histologically. Nine lines (two females and three males for heterozygotes only and three females and one male for homozygotes only) had only one sex evaluated.

10.1371/journal.pone.0180682.t001

###### KOMP lines evaluated.

Clinical summary of 169 KOMP strains screened for skin, hair, and nail abnormalities.

![](pone.0180682.t001){#pone.0180682.t001g}

  No.         Strain                                   JR \#                                    Mutant Allele                            No. Mice           Age (days)         Genotype   Skin        Hair Follicle   Seba-ceous Gland   Nail   Prenatal Lethality   Sub-viable
  ----------- ---------------------------------------- ---------------------------------------- ---------------------------------------- ------------------ ------------------ ---------- ----------- --------------- ------------------ ------ -------------------- ------------
  1           B6N(Cg)                                  24041                                    *4933427D14Rik*^*tm1*.*1(KOMP)Vlcg*^/J   2                  138/161            HET                                                                                   
  B6N(Cg)     24041                                    *4933427D14Rik*^*tm1*.*1(KOMP)Vlcg*^/J   2                                        138/161            HET                                                                                                      
  2           B6N(Cg)                                  24902                                    *Acsf2*^*tm1*.*1(KOMP)Vlcg*^/J           1                  218                HOM                                                                                   
  B6N(Cg)     24902                                    *Acsf2*^*tm1*.*1(KOMP)Vlcg*^/J           3                                        218/222            HOM                                                                                                      
  3           C57BL/6NJ                                25814                                    *Adad1*^*em1J*^/J                        2                  272/325            HET                                                                                   X
  C57BL/6NJ   25814                                    *Adad1*^*em1J*^/J                        2                                        308/337            HET                                                                                 X                    
  4           C57BL/6NJ                                25475                                    *Adad2*^*em1J*^/J                        2                  193/199            HET                                                                                   
  C57BL/6NJ   25475                                    *Adad2*^*em1J*^/J                        2                                        196/199            HET                                                                                                      
  5           B6N(Cg)                                  18592                                    *Adora2b*^*tm1*.*1(KOMP)Vlcg*^/J         2                  282                HOM                                                                                   
  B6N(Cg)     18592                                    *Adora2b*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        165/223            HET                                                                                                      
  6           B6N(Cg)                                  18561                                    *Ahrr*^*tm1b(KOMP)Wtsi*^/J               2                  41/138             HOM                                                                                   
  B6N(Cg)     18561                                    *Ahrr*^*tm1b(KOMP)Wtsi*^/J               2                                        41/138             HOM                                                                                                      
  7           B6N(Cg)                                  25117                                    *Akip1*^*tm1*.*1(KOMP)Vlcg*^/J           2                  183 /189           HOM                                                                                   
  B6N(Cg)     25117                                    *Akip1*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        183/189            HOM                                                                                                      
  8           B6N(Cg)                                  24334                                    *Akr1b8*^*tm1*.*1(KOMP)Vlcg*^/J          2                  125                HOM                                                                                   
  B6N(Cg)     24334                                    *Akr1b8*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        125                HOM                                                                                                      
  9           B6N(Cg)                                  24656                                    *Anapc15*^*tm1*.*1(KOMP)Vlcg*^/J         2                  125/139            HET                                                              X                    
  B6N(Cg)     24656                                    *Anapc15*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        166                HET                                                                          X                           
  10          B6N(Cg)                                  21510                                    *Ap4e1*^*tm1b(KOMP)Wtsi*^/J              2                  266                HOM                                                                                   
  B6N(Cg)     21510                                    *Ap4e1*^*tm1b(KOMP)Wtsi*^/J              2                                        198/359            HOM                                                                                                      
  11          B6N(Cg)                                  22304                                    *Arhgef10*^*tm1*.*1(KOMP)Vlcg*^/J        1                  95                 HOM                                                                                   
  B6N(Cg)     22304                                    *Arhgef10*^*tm1*.*1(KOMP)Vlcg*^/J        3                                        95/99              HOM                                                                                                      
  12          B6N(Cg)                                  18607                                    *Arrdc1*^*tm1*.*1(KOMP)Vlcg*^/J          3                  180/213            HET                                                                                   
  B6N(Cg)     18607                                    *Arrdc1*^*tm1*.*1(KOMP)Vlcg*^/J          1                                        180                HET                                                                                                      
  13          B6N(Cg)                                  18657                                    *Arsk*^*tm1b(KOMP)Wtsi*^/J               2                  254                HOM                                                                                   
  B6N(Cg)     18657                                    *Arsk*^*tm1b(KOMP)Wtsi*^/J               1                                        254                HOM                                                                                                      
  14          B6N(Cg)                                  18660                                    *Asb10*^*tm2b(KOMP)Wtsi*^/J              2                  173                HOM                                                                                   
  B6N(Cg)     18660                                    *Asb10*^*tm2b(KOMP)Wtsi*^/J              2                                        120                HOM                                                                                                      
  15          B6N(Cg)                                  22101                                    *Asf1a*^*tm1b(KOMP)Wtsi*^/2J             2                  320/385            HET                                                              X                    
  B6N(Cg)     22101                                    *Asf1a*^*tm1b(KOMP)Wtsi*^/2J             2                                        362/386            HET                                                                          X                           
  16          B6N(Cg)                                  22305                                    *Atrip*^*tm1b(KOMP)Wtsi*^/J              2                  340                HET                                                              X                    
  B6N(Cg)     22305                                    *Atrip*^*tm1b(KOMP)Wtsi*^/J              2                                        346                HET                                                                          X                           
  17          B6N(Cg)                                  24979                                    *Bbs7*^*tm1b(EUCOMM)Wtsi*^/J             2                  191/192            HET                                                                                   X
  B6N(Cg)     24979                                    *Bbs7*^*tm1b(EUCOMM)Wtsi*^/J             2                                        177/192            HET                                                                                 X                    
  18          B6N(Cg)                                  21751                                    *Btg2*^*tm1b(KOMP)Mbp*^/2J               2                  223                HOM                                                                                   
  B6N(Cg)     21751                                    *Btg2*^*tm1b(KOMP)Mbp*^/2J               2                                        204/352            HOM                                                                                                      
  19          B6N(Cg)                                  18653                                    *Bzw2*^*tm1b(KOMP)Mpb*^/J                2                  186                HOM                                                                                   
  B6N(Cg)     18653                                    *Bzw2*^*tm1b(KOMP)Mpb*^/J                2                                        226                HOM                                                                                                      
  20          B6N(Cg)                                  22307                                    *C1qa*^*tm1b(EUCOMM)Wtsi*^/3J            2                  308                HOM                                                                                   
  B6N(Cg)     22307                                    *C1qa*^*tm1b(EUCOMM)Wtsi*^/3J            2                                        308                HOM                                                                                                      
  21          B6N(Cg)                                  18562                                    *C1qtnf5*^*tm1*.*1(KOMP)Vlcg*^/J         2                  380/472            HOM                                                                                   
  B6N(Cg)     18562                                    *C1qtnf5*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        443                HOM                                                                                                      
  22          B6N(Cg)                                  22098                                    *C3*^*tm1*.*1(KOMP)Vlcg*^/J              2                  128/219            HOM                                                                                   
  B6N(Cg)     22098                                    *C3*^*tm1*.*1(KOMP)Vlcg*^/J              2                                        128/225            HOM                                                                                                      
  23          B6N(Cg)                                  21282                                    *C9*^*tm1(KOMP)Vlcg*^/J                  1                  198/206            HET                                                                                   
  B6N(Cg)     21282                                    *C9*^*tm1(KOMP)Vlcg*^/J                  2                                        269                HET                                                                                                      
  24          B6N(Cg)                                  22418                                    *Cacna2d3*^*tm1b(KOMP)Wtsi*^/J           2                  239                HOM                                                                                   
  B6N(Cg)     22418                                    *Cacna2d3*^*tm1b(KOMP)Wtsi*^/J           2                                        239                HOM                                                                                                      
  25          B6N(Cg)                                  23393                                    *Cast*^*tm1*.*1(KOMP)Vlcg*^/2J           2                  111/171            HOM                                                                                   
  B6N(Cg)     23393                                    *Cast*^*tm1*.*1(KOMP)Vlcg*^/2J           2                                        111/171            HOM                                                                                                      
  26          B6N(Cg)                                  18111                                    *Ccd20*^*tm1(KOMP)Vlcg*^/J               2                  337/339            HET                                                              X                    
  27          B6N(Cg)                                  24349                                    *Ccl26*^*tm1*.*1(KOMP)Vlcg*^/J           3                  216/232/ 251       HOM                                                                                   
  B6N(Cg)     24349                                    *Ccl26*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        157                HOM                                                                                                      
  28          B6N(Cg)                                  22016                                    *Cd33*^*tm1*.*1(KOMP)Vlcg*^/J            4                  147/321            HOM                                                                                   
  B6N(Cg)     22016                                    *Cd33*^*tm1*.*1(KOMP)Vlcg*^/J            4                                        176/321            HOM                                                                                                      
  29          B6N(Cg)                                  22335                                    *Ceacam16*^*tm1*.*1(KOMP)Wtsi*^/J        4                  100/207/ 227/285   HOM                                                                                   
  B6N(Cg)     22335                                    *Ceacam16*^*tm1*.*1(KOMP)Wtsi*^/J        4                                        100/207/ 273/285   HOM                                                                                                      
  30          B6N(Cg)                                  18836                                    *Cenpo*^*tm1b(KOMP)Mbp*^/J               2                  313/379            HET                                                              X                    
  B6N(Cg)     18836                                    *Cenpo*^*tm1b(KOMP)Mbp*^/J               2                                        354/379            HET                                                                          X                           
  31          B6N(Cg)                                  22819                                    *Cers5*^*tm2b(KOMP)Mbp*^/J               2                  206                HOM                                                                                   
  B6N(Cg)     22819                                    *Cers5*^*tm2b(KOMP)Mbp*^/J               2                                        206                HOM                                                                                                      
  32          B6N(Cg)                                  18603                                    *Chn1*^*tm1*.*1(KOMP)Vlcg*^/J            2                  244/247            HOM                                                                                   
  B6N(Cg)     18603                                    *Chn1*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        244/246            HOM                                                                                                      
  33          B6N(Cg)                                  25118                                    *Chp2*^*tm1b(KOMP)Mbp*^/J                3                  149/210/ 211       HET                                                                                   
  B6N(Cg)     25118                                    *Chp2*^*tm1b(KOMP)Mbp*^/J                2                                        210/211            HET                                                                                                      
  34          B6N(Cg)                                  23317                                    *Chtf18*^*tm1b(KOMP)Wtsi*^/J             2                  300                HET                                                                                   X
  B6N(Cg)     23317                                    *Chtf18*^*tm1b(KOMP)Wtsi*^/J             2                                        300                HET                                                                                 X                    
  35          B6N(Cg)                                  18631                                    *Cinp*^*tm1*.*1(KOMP)Vlcg*^/J            2                  126/136            HET                                                              X                    
  B6N(Cg)     18631                                    *Cinp*^*tm1*.*1(KOMP)Vlcg*^/J            4                                        131/135/ 136       HET                                                                          X                           
  36          B6N(Cg)                                  18587                                    *Cited4*^*tm1*.*1(KOMP)Vlcg*^/J          2                  140/183            HOM                                                                                   
  B6N(Cg)     18587                                    *Cited4*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        140/183            HOM                                                                                                      
  37          B6N(Cg)                                  23666                                    *Cldn19*^*tm1*.*1(KOMP)Vlcg*^/J          2                  230/324            HOM                                                                                   
  B6N(Cg)     23666                                    *Cldn19*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        230/255            HOM                                                                                                      
  38a         B6N(Cg)                                  18604                                    *Col18a1*^*tm1*.*1(KOMP)Vlcg*^/J         2                  193/208            HET                                                                                   
  B6N(Cg)     18604                                    *Col18a1*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        191/208            HET                                                                                                      
  38b         B6N(Cg)                                  18604                                    *Col18a1*^*tm1*.*1(KOMP)Vlcg*^/J         2                  322/350            HOM                                                                                   
  B6N(Cg)     18604                                    *Col18a1*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        322/350            HOM                                                                                                      
  39          B6N(Cg)                                  21485                                    *Cp*^*tm1b(KOMP)Wtsi*^/J                 2                  184/326            HOM                                                                                   
  B6N(Cg)     21485                                    *Cp*^*tm1b(KOMP)Wtsi*^/J                 2                                        271/326            HOM                                                                                                      
  40          B6N(Cg)                                  22015                                    *Crym*^*tm1b(KOMP)Wtsi*^/J               2                  161/198            HOM                                                                                   
  B6N(Cg)     22015                                    *Crym*^*tm1b(KOMP)Wtsi*^/J               2                                        161/198            HOM                                                                                                      
  41          B6N(Cg)                                  22096                                    *Cyb5d1*^*tm1*.*1(KOMP)Vlcg*^/J          3                  323/354            HOM                                                                                   
  B6N(Cg)     22096                                    *Cyb5d1*^*tm1*.*1(KOMP)Vlcg*^/J          1                                        355                HOM                                                                                                      
  42          B6N(Cg)                                  24035                                    *Cyb5d2*^*tm1*.*1(KOMP)Wtsi*^/J          2                  203                HOM                                                                                   
  B6N(Cg)     24035                                    *Cyb5d2*^*tm1*.*1(KOMP)Wtsi*^/J          2                                        203                HOM                                                                                                      
  43          B6N(Cg)                                  22777                                    *Dbf4*^*tm1b(KOMP)Wtsi*^/J               2                  137/155            HET                                                              X                    
  B6N(Cg)     22777                                    *Dbf4*^*tm1b(KOMP)Wtsi*^/J               2                                        178                HET                                                                          X                           
  44          B6N(Cg)                                  27202                                    *Dbn1*^*tm1a(KOMP)Wtsi*^/J               2                  274                HET                                                                                   X
  B6N(Cg)     27202                                    *Dbn1*^*tm1a(KOMP)Wtsi*^/J               2                                        200/241            HET                                                                                 X                    
  45          B6N(Cg)                                  23604                                    *Dcps*^*tm1b(EUCOMM)Hmgu*^/J             2                  249                HET                                                              X                    
  B6N(Cg)     23604                                    *Dcps*^*tm1b(EUCOMM)Hmgu*^/J             2                                        258                HET                                                                          X                           
  46          B6N(Cg)                                  18632                                    *Dnajb3*^*tm1*.*1(KOMP)Vlcg*^/J          2                  209/240            HOM                                                                                   
  B6N(Cg)     18632                                    *Dnajb3*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        202/240            HOM                                                                                                      
  47          B6N(Cg)                                  18580                                    *Dnajb7*^*tm1*.*1(KOMP)Vlcg*^/J          1                  250                HOM                                                                                   
  B6N(Cg)     18580                                    *Dnajb7*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        268                HOM                                                                                                      
  48          B6N(Cg)                                  18597                                    *Dnajb12*^*tm1*.*1(KOMP)Vlcg*^/J         2                  156/243            HOM                                                                                   
  49          B6N(Cg)                                  18616                                    *Dnajc5g*^*tm1*.*1(KOMP)Vlcg*^/J         2                  301                HOM                                                                                   
  B6N(Cg)     18616                                    *Dnajc5g*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        164                HOM                                                                                                      
  50          B6N(Cg)                                  18637                                    *Dnajc14*^*tm1*.*1(KOMP)Vlcg*^/J         4                  136/177            HOM                                                                                   
  B6N(Cg)     18637                                    *Dnajc14*^*tm1*.*1(KOMP)Vlcg*^/J         4                                        117/177/ 352       HOM                                                                                                      
  51          B6N(Cg)                                  18633                                    *Dnajc16*^*tm1*.*1(KOMP)Vlcg*^/J         1                  122                HET                                                                                   X
  B6N(Cg)     18633                                    *Dnajc16*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        122                HET                                                                                 X                    
  52          B6N(Cg)                                  21508                                    *Dnase1l2*^*tm1*.*1(KOMP)Wtsi*^/J        2                  326                HOM                                                                                   
  53          B6N(Cg)                                  26437                                    *Enox1*^*tm1*.*1(KOMP)Vlcg*^/J           3                  239                HET                                                                                   
  B6N(Cg)     26437                                    *Enox1*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        266                HET                                                                                                      
  54          B6N(Cg)                                  23489                                    *Ermp1*^*tm1*.*1(KOMP)Vlcg*^/J           4                  167/201/ 220       HOM                                                                                   
  B6N(Cg)     23489                                    *Ermp1*^*tm1*.*1(KOMP)Vlcg*^/J           4                                        170/173/ 201       HOM                                                                                                      
  55          B6N(Cg)                                  24660                                    *Espnl*^*tm1b(KOMP)Wtsi*^/2J             2                  180                HOM                                                                                   
  B6N(Cg)     24660                                    *Espnl*^*tm1b(KOMP)Wtsi*^/2J             2                                        145                HOM                                                                                                      
  56          C57BL/6NJ                                25733                                    *Exoc4*^*em1J*^/J                        4                  121/123            HET                                                              X                    
  C57BL/6NJ   25733                                    *Exoc4*^*em1J*^/J                        2                                        121/123            HET                                                                          X                           
  57          B6N(Cg)                                  18622                                    *Exosc8*^*tm1*.*1(KOMP)Vlcg*^/J          2                  379                HET                                                              X                    
  B6N(Cg)     18622                                    *Exosc8*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        429                HET                                                                          X                           
  58          B6N(Cg)                                  18705                                    *Fam161a*^*tm1b(KOMP)Wtsi*^/2J           2                  209                HOM                                                                                   
  B6N(Cg)     18705                                    *Fam161a*^*tm1b(KOMP)Wtsi*^/2J           2                                        253/316            HOM                                                                                                      
  59          B6N(Cg)                                  22011                                    *Far2*^*tm2b(KOMP)Wtsi*^/2J              2                  184                HOM                    dystrophy       hypo-plasia                                    
  B6N(Cg)     22011                                    *Far2*^*tm2b(KOMP)Wtsi*^/2J              2                                        172/184            HOM                           dystrophy   hypo-plasia                                                    
  60          B6N(Cg)                                  22097                                    *Foxo3*^*tm1*.*1(KOMP)Vlcg*^/J           2                  148                HOM                                                                                   
  B6N(Cg)     22097                                    *Foxo3*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        226                HOM                                                                                                      
  61          B6N(Cg)                                  21486                                    *Ghr*^*tm1b(KOMP)Wtsi*^/3J               2                  304/337            HET                                                                                   
  B6N(Cg)     21486                                    *Ghr*^*tm1b(KOMP)Wtsi*^/3J               2                                        304/337            HET                                                                                                      
  62          B6N(Cg)                                  19459                                    *Ghrhr*^*tm1*.*1(KOMP)Vlcg*^/2J          1                  292                HOM                                                                                   
  B6N(Cg)     19459                                    *Ghrhr*^*tm1*.*1(KOMP)Vlcg*^/2J          2                                        141                HOM                                                                                                      
  63          B6N(Cg)                                  18595                                    *Ghsr*^*tm1*.*1(KOMP)Vlcg*^/J            4                  143/153/ 370       HOM                                                                                   
  B6N(Cg)     18595                                    *Ghsr*^*tm1*.*1(KOMP)Vlcg*^/J            4                                        156/370            HOM                                                                                                      
  64          B6N(Cg)                                  18643                                    *Gipc3*^*tm1b(KOMP)Wtsi*^/J              2                  208                HOM                                                                                   
  B6N(Cg)     18643                                    *Gipc3*^*tm1b(KOMP)Wtsi*^/J              2                                        208                HOM                                                                                                      
  65          B6N(Cg)                                  18558                                    *Glycam1*^*tm1*.*1(KOMP)Vlcg*^/J         1                  180                HOM                                                                                   
  B6N(Cg)     18558                                    *Glycam1*^*tm1*.*1(KOMP)Vlcg*^/J         1                                        180                HOM                                                                                                      
  66          B6N(Cg)                                  24058                                    *Gnb5*^*tm1*.*1(KOMP)Vlcg*^/J            2                  174/185            HET                                                                                   X
  B6N(Cg)     24058                                    *Gnb5*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        186                HET                                                                                 X                    
  67          B6N(Cg)                                  18594                                    *H1fx*^*tm1*.*1(KOMP)Vlcg*^/J            2                  189                HOM                                                                                   
  B6N(Cg)     18594                                    *H1fx*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        186/195            HOM                                                                                                      
  68          C57BL/6NJ                                25319                                    *Heyl*^*tm1b(KOMP)Wtsi*^/J               2                  215                HOM                                                                                   
  C57BL/6NJ   25319                                    *Heyl*^*tm1b(KOMP)Wtsi*^/J               2                                        215                HOM                                                                                                      
  69          B6N(Cg)                                  24947                                    *Hfe2*^*tm1b(KOMP)Wtsi*^/J               2                  168/197            HOM                                                                                   
  B6N(Cg)     24947                                    *Hfe2*^*tm1b(KOMP)Wtsi*^/J               2                                        197/198            HOM                                                                                                      
  70          B6N(Cg)                                  24981                                    *Hsf2*^*tm1b(KOMP)Wtsi*^/3J              2                  172                HOM                                                                                   
  B6N(Cg)     24981                                    *Hsf2*^*tm1b(KOMP)Wtsi*^/3J              2                                        172                HOM                                                                                                      
  71          B6N(Cg)                                  22782                                    *Hspb1*^*tm1(KOMP)Vlcg*^/2J              2                  188                HET                                                                                   
  72          B6N(Cg)                                  18619                                    *Hspb2*^*tm1*.*1(KOMP)Vlcg*^/J           4                  262/263/ 386       HOM                                                                                   
  B6N(Cg)     18619                                    *Hspb2*^*tm1*.*1(KOMP)Vlcg*^/J           4                                        262/263/ 386       HOM                                                                                                      
  73          B6N(Cg)                                  18618                                    *Hspb3*^*tm1*.*1(KOMP)Vlcg*^/J           2                  144/199            HOM                                                                                   
  B6N(Cg)     18618                                    *Hspb3*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        144/199            HOM                                                                                                      
  74          B6N(Cg)                                  18617                                    *Htr1a*^*tm1*.*1(KOMP)Vlcg*^/J           2                  345                HOM                                                                                   
  B6N(Cg)     18617                                    *Htr1a*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        137/308            HOM                                                                                                      
  75          B6N(Cg)                                  24347                                    *Htr1d*^*tm1*.*1(KOMP)Vlcg*^/2J          2                  165                HOM                                                                                   
  B6N(Cg)     24347                                    *Htr1d*^*tm1*.*1(KOMP)Vlcg*^/2J          2                                        165/226            HOM                                                                                                      
  76          B6N(Cg)                                  18615                                    *Htr3b*^*tm1*.*1(KOMP)Vlcg*^/J           2                  299                HOM                                                                                   
  B6N(Cg)     18615                                    *Htr3b*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        299                HOM                                                                                                      
  77          B6N(Cg)                                  23606                                    *Ift88*^*tm1*.*1(KOMP)Vlcg*^/J           2                  350                HET                                                              X                    
  B6N(Cg)     23606                                    *Ift88*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        349/350            HET                                                                          X                           
  78a         B6N(Cg)                                  21516                                    *Igsf11*^*tm1b(KOMP)Wtsi*^/J             1                  297                HET                                                                                   
  B6N(Cg)     21516                                    *Igsf11*^*tm1b(KOMP)Wtsi*^/J             3                                        287/300            HET                                                                                                      
  78b         B6N(Cg)                                  21516                                    *Igsf11*^*tm1b(KOMP)Wtsi*^/J             2                  280/351            HOM                                                                                   
  B6N(Cg)     21516                                    *Igsf11*^*tm1b(KOMP)Wtsi*^/J             2                                        280                HOM                                                                                                      
  79          B6N(Cg)                                  22303                                    *Il12rb2*^*tm1*.*1(KOMP)Vlcg*^/J         4                  168/206/ 264       HOM                                                                                   
  B6N(Cg)     22303                                    *Il12rb2*^*tm1*.*1(KOMP)Vlcg*^/J         4                                        168/206            HOM                                                                                                      
  80          B6N(Cg)                                  22005                                    *Il24*^*tm1*.*1(KOMP)Vlcg*^/2J           4                  64/165/194/214     HOM                                                                                   
  B6N(Cg)     22005                                    *Il24*^*tm1*.*1(KOMP)Vlcg*^/2J           4                                        64/165/194/214     HOM                                                                                                      
  81          B6N(Cg)                                  22388                                    *Il6st*^*tm1b(KOMP)Mbp*^/J               2                  180/210            HOM                                                                                   
  B6N(Cg)     22388                                    *Il6st*^*tm1b(KOMP)Mbp*^/J               2                                        210                HOM                                                                                                      
  82          B6N(Cg)                                  18655                                    *Irf8*^*tm1b(KOMP)Wtsi*^/J               2                  195                HOM                                                                                   
  B6N(Cg)     18655                                    *Irf8*^*tm1b(KOMP)Wtsi*^/J               2                                        171/195            HOM                                                                                                      
  83          B6N(Cg)                                  24055                                    *Jam2*^*tm1*.*1(KOMP)Mbp*^/J             2                  140                HOM                                                                                   
  B6N(Cg)     24055                                    *Jam2*^*tm1*.*1(KOMP)Mbp*^/J             2                                        140                HOM                                                                                                      
  84          B6N(Cg)                                  26066                                    *Jmjd8*^*tm1*.*1(KOMP)Vlcg*^/J           3                  269                HOM                                                                                   
  B6N(Cg)     26066                                    *Jmjd8*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        269                HOM                                                                                                      
  85          B6N(Cg)                                  22821                                    *Jup*^*tm1*.*1(KOMP)Vlcg*^/J             2                  109/143            HET                                                              X                    
  B6N(Cg)     22821                                    *Jup*^*tm1*.*1(KOMP)Vlcg*^/J             2                                        107/143            HET                                                                          X                           
  86          B6N(Cg)                                  21140                                    *Kcnh3*^*tm1*.*1(KOMP)Vlcg*^/2J          1                  186                HOM                                                                                   
  B6N(Cg)     21140                                    *Kcnh3*^*tm1*.*1(KOMP)Vlcg*^/2J          2                                        186/288            HOM                                                                                                      
  87          B6N(Cg)                                  23603                                    *Kif1b*^*tm1b(KOMP)Wtsi*^/2J             2                  319                HET                                                              X                    
  B6N(Cg)     23603                                    *Kif1b*^*tm1b(KOMP)Wtsi*^/2J             2                                        322                HET                                                                          X                           
  88          B6N(Cg)                                  22085                                    *Kif26b*^*tm2b(KOMP)Wtsi*^/J             2                  323/446            HET                                                              X                    
  B6N(Cg)     22085                                    *Kif26b*^*tm2b(KOMP)Wtsi*^/J             2                                        327/446            HET                                                                          X                           
  89          B6N(Cg)                                  25381                                    *Klk14*^*tm1*.*1(KOMP)Vlcg*^/J           2                  115/161            HOM                                                                                   
  B6N(Cg)     25381                                    *Klk14*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        115/152            HOM                                                                                                      
  90          B6N(Cg)                                  23318                                    *Krt17*^*tm1*.*1(KOMP)Vlcg*^/2J          2                  297/304            HET                                                                                   
  B6N(Cg)     23318                                    *Krt17*^*tm1*.*1(KOMP)Vlcg*^/2J          2                                        297/304            HET                                                                                                      
  91          B6N(Cg)                                  25383                                    *Krt9*^*tm1*.*1(KOMP)Vlcg*^/J            2                  210                HOM                                                                                   
  B6N(Cg)     25383                                    *Krt9*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        210                HOM                                                                                                      
  92          B6N(Cg)                                  25518                                    *Lipn*^*tm1*.*1(KOMP)Vlcg*^/J            4                  188/189/ 195       HOM                                                                                   
  93          B6N(Cg)                                  18563                                    *Loxl1*^*tm1*.*1(KOMP)Vlcg*^/J           4                  112/133/ 147       HET                                                                                   
  B6N(Cg)     18563                                    *Loxl1*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        110                HET                                                                                                      
  94          B6N(Cg)                                  22094                                    *Lpar6*^*tm1*.*1(KOMP)Vlcg*^/J           2                  172/226            HOM                                                                                   
  B6N(Cg)     22094                                    *Lpar6*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        172/213            HOM                                                                                                      
  95          B6N(Cg)                                  23021                                    *Lrrc15*^*tm1b(KOMP)Wtsi*^/J             1                  183                HOM                                                                                   
  B6N(Cg)     23021                                    *Lrrc15*^*tm1b(KOMP)Wtsi*^/J             1                                        48                 HOM                                                                                                      
  96          B6N(Cg)                                  23024                                    *Mdk*^*tm1*.*1(KOMP)Mbp*^/J              2                  193                HOM                                                                                   
  B6N(Cg)     23024                                    *Mdk*^*tm1*.*1(KOMP)Mbp*^/J              2                                        193                HOM                                                                                                      
  97a         B6N(Cg)                                  22092                                    *Mmp8*^*tm1(KOMP)Vlcg*^/J                2                  213                HET                                                                                   
  97b         B6N(Cg)                                  22092                                    *Mmp8*^*tm1*.*1(KOMP)Vlcg*^/J            2                  232/273            HOM                                                                                   
  B6N(Cg)     22092                                    *Mmp8*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        245/273            HOM                                                                                                      
  98          B6N(Cg)                                  18635                                    *Mrps25*^*tm1*.*1(KOMP)Vlcg*^/J          2                  303                HET                                                              X                    
  B6N(Cg)     18635                                    *Mrps25*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        303/364            HET                                                                          X                           
  99          B6N(Cg)                                  24673                                    *Myo7b*^*tm1b(EUCOMM)Hmgu*^/J            2                  237/240            HOM                                                                                   
  B6N(Cg)     24673                                    *Myo7b*^*tm1b(EUCOMM)Hmgu*^/J            2                                        236/237            HOM                                                                                                      
  100         B6N(Cg)                                  23599                                    *Nat1*^*tm1*.*1(KOMP)Vlcg*^/J            2                  142/152            HOM                                                                                   
  B6N(Cg)     23599                                    *Nat1*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        142/157            HOM                                                                                                      
  101         B6N(Cg)                                  18575                                    *Ncald*^*tm1*.*1(KOMP)Vlcg*^/J           3                  230/315            HOM                                                                                   
  B6N(Cg)     18575                                    *Ncald*^*tm1*.*1(KOMP)Vlcg*^/J           3                                        230/310/ 408       HOM                                                                                                      
  102         B6N(Cg)                                  18577                                    *Nefh*^*tm1*.*1(KOMP)Vlcg*^/J            1                  230                HOM                                                                                   
  B6N(Cg)     18577                                    *Nefh*^*tm1*.*1(KOMP)Vlcg*^/J            1                                        230                HOM                                                                                                      
  103         B6N(Cg)                                  18697                                    *Nemf*^*tm1b(EUCOMM)Hmgu*^/J             2                  78                 HET                                                              X                    
  B6N(Cg)     18697                                    *Nemf*^*tm1b(EUCOMM)Hmgu*^/J             2                                        78                 HET                                                                          X                           
  104         B6N(Cg)                                  24185                                    *Nes*^*tm1b(KOMP)Wtsi*^/J                2                  139/140            HOM                                                                                   
  B6N(Cg)     24185                                    *Nes*^*tm1b(KOMP)Wtsi*^/J                2                                        162/170            HOM                                                                                                      
  105         B6N(Cg)                                  18638                                    *Nmrk2*^*tm1*.*1(KOMP)Vlcg*^/J           2                  172/177            HOM                                                                                   
  B6N(Cg)     18638                                    *Nmrk2*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        177/180            HOM                                                                                                      
  106         B6N(Cg)                                  18645                                    *Nrcam*^*tm2e*.*1(KOMP)Wtsi*^/J          2                  242/275            HOM                                                                                   
  B6N(Cg)     18645                                    *Nrcam*^*tm2e*.*1(KOMP)Wtsi*^/J          2                                        242/275            HOM                                                                                                      
  107         B6N(Cg)                                  24899                                    *Nsun7*^*tm1*.*1(KOMP)Vlcg*^/J           3                  183/190            HET                                                                                   
  B6N(Cg)     24899                                    *Nsun7*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        183                HET                                                                                                      
  108         B6N(Cg)                                  24233                                    *Nxn*^*tm1b(EUCOMM)Wtsi*^/J              2                  232/236            HET                                                              X                    
  B6N(Cg)     24233                                    *Nxn*^*tm1b(EUCOMM)Wtsi*^/J              2                                        236                HET                                                                          X                           
  109         B6N(Cg)                                  24036                                    *Orc1*^*tm1b(KOMP)Wtsi*^/J               2                  185                HET                                                              X                    
  B6N(Cg)     24036                                    *Orc1*^*tm1b(KOMP)Wtsi*^/J               2                                        185/206            HET                                                                          X                           
  110         B6N(Cg)                                  22338                                    *Osm*^*tm1b(KOMP)Wtsi*^/J                4                  198/270/ 358/376   HOM                                                                                   
  B6N(Cg)     22338                                    *Osm*^*tm1b(KOMP)Wtsi*^/J                4                                        198/270/ 358       HOM                                                                                                      
  111         B6N(Cg)                                  23058                                    *Parp8*^*tm1*.*1(KOMP)Wtsi*^/J           2                  202                HOM                                                                                   
  B6N(Cg)     23058                                    *Parp8*^*tm1*.*1(KOMP)Wtsi*^/J           2                                        202                HOM                                                                                                      
  112         B6N(Cg)                                  21613                                    *Parp16*^*tm1*.*1(KOMP)Mbp*^/2J          2                  288                HOM                                                                                   
  B6N(Cg)     21613                                    *Parp16*^*tm1*.*1(KOMP)Mbp*^/2J          2                                        265                HOM                                                                                                      
  113         B6N(Cg)                                  22341                                    *Pcsk5*^*tm1b(KOMP)Mbp*^/J               2                  306/321            HET                                                              X                    
  B6N(Cg)     22341                                    *Pcsk5*^*tm1b(KOMP)Mbp*^/J               2                                        346                HET                                                                          X                           
  114         B6N(Cg)                                  24330                                    *Pnmt*^*tm1*.*1(KOMP)Vlcg*^/J            4                  120/140/ 152/182   HOM                                                                                   
  B6N(Cg)     24330                                    *Pnmt*^*tm1*.*1(KOMP)Vlcg*^/J            3                                        140/182            HOM                                                                                                      
  115a        B6N(Cg)                                  18609                                    *Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J         4                  139/282            HET                    vibrissa                                                       
  B6N(Cg)     18609                                    *Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        139/330            HET                           vibrissa                                                                   
  115b        B6N(Cg)                                  18609                                    *Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J         2                  321                HOM                                                                                   
  B6N(Cg)     18609                                    *Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        114/137            HOM                                                                                                      
  116         B6N(Cg)                                  22099                                    *Prep*^*tm1b(KOMP)Wtsi*^/J               2                  328/350            HET                                                              X                    
  B6N(Cg)     22099                                    *Prep*^*tm1b(KOMP)Wtsi*^/J               2                                        350                HET                                                                          X                           
  117a        B6N(Cg)                                  18576                                    *Prom2*^*tm1*.*1(KOMP)Vlcg*^/J           3                  89                 HET                                                                                   
  B6N(Cg)     18576                                    *Prom2*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        89                 HET                                                                                                      
  117b        B6N(Cg)                                  18576                                    *Prom2*^*tm1*.*1(KOMP)Vlcg*^/J           2                  403                HOM                                                                                   
  B6N(Cg)     18576                                    *Prom2*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        437                HOM                                                                                                      
  118         B6N(Cg)                                  24025                                    *Psen1*^*tm1*.*1(KOMP)Vlcg*^/J           2                  225/240            HET                                                              X                    
  B6N(Cg)     24025                                    *Psen1*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        240                HET                                                                          X                           
  119         B6N(Cg)                                  23667                                    *Pycr1*^*tm1*.*1(KOMP)Vlcg*^/J           2                  165/251            HOM                                                                                   
  B6N(Cg)     23667                                    *Pycr1*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        165/251            HOM                                                                                                      
  120         B6N(Cg)                                  22387                                    *Rab5a*^*tm1b(KOMP)Wtsi*^/J              4                  222/230            HOM                                                                                   
  B6N(Cg)     22387                                    *Rab5a*^*tm1b(KOMP)Wtsi*^/J              4                                        222/227/ 230       HOM                                                                                                      
  121         B6N(Cg)                                  22389                                    *Rab24*^*tm1(KOMP)Wtsi*^/J               2                  179                HET                                                                                   
  B6N(Cg)     22389                                    *Rab24*^*tm1(KOMP)Wtsi*^/J               2                                        243                HET                                                                                                      
  122         B6N(Cg)                                  23422                                    *Rab27b*^*tm1b(KOMP)Wtsi*^/J             2                  127/129            HOM                                                                                   
  B6N(Cg)     23422                                    *Rab27b*^*tm1b(KOMP)Wtsi*^/J             2                                        133/159            HOM                                                                                                      
  123         B6N(Cg)                                  24188                                    *Rab34*^*tm1b(EUCOMM)Hmgu*^/J            2                  328                HET                                                              X                    
  B6N(Cg)     24188                                    *Rab34*^*tm1b(EUCOMM)Hmgu*^/J            2                                        328                HET                                                                          X                           
  124         B6N(Cg)                                  23664                                    *Rab36*^*tm1b(KOMP)Wtsi*^/J              2                  96/255             HOM                                                                                   
  B6N(Cg)     23664                                    *Rab36*^*tm1b(KOMP)Wtsi*^/J              2                                        96/255             HOM                                                                                                      
  125         B6N(Cg)                                  23059                                    *Rab43*^*tm1*.*1(KOMP)Vlcg*^/J           2                  130                HOM                                                                                   
  B6N(Cg)     23059                                    *Rab43*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        130                HOM                                                                                                      
  126         B6N(Cg)                                  26882                                    *Rabac1*^*tm1*.*1(KOMP)Vlcg*^/J          2                  118                HOM                                                                                   
  B6N(Cg)     26882                                    *Rabac1*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        118                HOM                                                                                                      
  127         B6N(Cg)                                  25849                                    *Rbpj*^*tm1b(EUCOMM)Hmgu*^/J             2                  298                HET                                                              X                    
  B6N(Cg)     25849                                    *Rbpj*^*tm1b(EUCOMM)Hmgu*^/J             2                                        255                HET                                                                          X                           
  128         B6N(Cg)                                  18572                                    *Resp18*^*tm1*.*1(KOMP)Vlcg*^/J          1                  231                HOM                                                                                   
  B6N(Cg)     18572                                    *Resp18*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        231                HOM                                                                                                      
  129         B6N(Cg)                                  24674                                    *Rhbdl2*^*tm1b(KOMP)Wtsi*^/J             2                  138                HOM                                                                                   
  B6N(Cg)     24674                                    *Rhbdl2*^*tm1b(KOMP)Wtsi*^/J             2                                        138                HOM                                                                                                      
  130         B6N(Cg)                                  21750                                    *Rilpl2*^*tm1b(KOMP)Wtsi*^/J             4                  150/198/ 287       HOM                                                                                   
  B6N(Cg)     21750                                    *Rilpl2*^*tm1b(KOMP)Wtsi*^/J             4                                        242/287/ 295       HOM                                                                                                      
  131         B6N(Cg)                                  21511                                    *Rnf10*^*tm1b(KOMP)Wtsi*^/J              2                  222                HOM                                                                                   
  B6N(Cg)     21511                                    *Rnf10*^*tm1b(KOMP)Wtsi*^/J              2                                        228                HOM                                                                                                      
  132         B6N(Cg)                                  26493                                    *Rps6kl1*^*tm1*.*1(KOMP)Vlcg*^/J         2                  242                HOM                                                                                   
  B6N(Cg)     26493                                    *Rsp6kl1*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        242                HOM                                                                                                      
  133         B6N(Cg)                                  23136                                    *Sdha*^*tm2b(KOMP)Wtsi*^/2J              2                  343/393            HET                                                              X                    
  B6N(Cg)     23136                                    *Sdha*^*tm2b(KOMP)Wtsi*^/2J              2                                        393/396            HET                                                                          X                           
  134         B6N(Cg)                                  24038                                    *Sgta*^*tm1b(KOMP)Mbp*^/J                2                  250/254            HET                                                                                   
  B6N(Cg)     24038                                    *Sgta*^*tm1b(KOMP)Mbp*^/J                2                                        250                HET                                                                                                      
  135         B6N(Cg)                                  21142                                    *Sh3tc2*^*tm1b(KOMP)Wtsi*^/J             2                  221                HOM                                                                                   
  B6N(Cg)     21142                                    *Sh3tc2*^*tm1b(KOMP)Wtsi*^/J             1                                        227                HOM                                                                                                      
  136         B6N(Cg)                                  23511                                    *Ska2*^*tm1b(KOMP)Wtsi*^/J               2                  228                HET                                                              X                    
  B6N(Cg)     23511                                    *Ska2*^*tm1b(KOMP)Wtsi*^/J               2                                        231                HET                                                                          X                           
  137         B6N(Cg)                                  23414                                    *Slc25a35*^*tm1*.*1(KOMP)Vlcg*^/J        2                  179                HOM                                                                                   
  B6N(Cg)     23414                                    *Slc25a35*^*tm1*.*1(KOMP)Vlcg*^/J        2                                        179                HOM                                                                                                      
  138a        B6N(Cg)                                  22780                                    *Sorbs2*^*tm1(KOMP)Mbp*^/J               1                  86                 HET                                                                                   
  B6N(Cg)     22780                                    *Sorbs2*^*tm1(KOMP)Mbp*^/J               3                                        86/199             HET                                                                                                      
  138b        B6N(Cg)                                  22780                                    *Sorbs2*^*tm1*.*1(KOMP)Mbp*^/J           1                  159                HOM                                                                                   
  B6N(Cg)     22780                                    *Sorbs2*^*tm1*.*1(KOMP)Mbp*^/J           2                                        159/160            HOM                                                                                                      
  139         B6N(Cg)                                  23020                                    *Sox18*^*tm1*.*1(KOMP)Vlcg*^/J           2                  178/226            HOM                                                                                   
  B6N(Cg)     23020                                    *Sox18*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        161/165            HOM                                                                                                      
  140         B6N(Cg)                                  26095                                    *Spag4*^*tm1*.*1(KOMP)Mbp*^/J            2                  161                HOM                                                                                   
  B6N(Cg)     26095                                    *Spag4*^*tm1*.*1(KOMP)Mbp*^/J            1                                        59                 HOM                                                                                                      
  141         B6N(Cg)                                  18820                                    *Sptssa*^*tm1b(KOMP)Wtsi*^/J             2                  287/292            HET                                                              X                    
  B6N(Cg)     18820                                    *Sptssa*^*tm1b(KOMP)Wtsi*^/J             2                                        327                HET                                                                          X                           
  142         B6N(Cg)                                  24184                                    *Srd5a3*^*tm1b(EUCOMM)Wtsi*^/J           2                  258/265            HET                                                              X                    
  B6N(Cg)     24184                                    *Srd5a3*^*tm1b(EUCOMM)Wtsi*^/J           2                                        265                HET                                                                          X                           
  143         B6N(Cg)                                  21115                                    *Stag3*^*tm1e*.*1(KOMP)Wtsi*^*/2*J       1                  234                HOM                                                                                   
  B6N(Cg)     21115                                    *Stag3*^*tm1e*.*1(KOMP)Wtsi*^/2J         2                                        104/275            HOM                                                                                                      
  144         B6N(Cg)                                  18611                                    *Stk16*^*tm1*.*1(KOMP)Vlcg*^/J           2                  105                HET                                                              X                    
  B6N(Cg)     18611                                    *Stk16*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        66/105             HET                                                                          X                           
  145         B6N(Cg)                                  18611                                    *Stk16*^*tm1*.*1(KOMP)Vlcg*^/J           2                  39                 HOM                                                                                   
  B6N(Cg)     18611                                    *Stk16*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        39                 HOM                                                                                                      
  146         B6N(Cg)                                  22781                                    *Stx16*^*tm1b(KOMP)Wtsi*^/J              1                  301                HOM                                                                                   
  B6N(Cg)     22781                                    *Stx16*^*tm1b(KOMP)Wtsi*^/J              2                                        301/320            HOM                                                                                                      
  147         B6N(Cg)                                  22784                                    *Sycp3*^*tm1(KOMP)Vlcg*^/J               2                  110                HET                                                                                   
  B6N(Cg)     22784                                    *Sycp3*^*tm1(KOMP)Vlcg*^/J               2                                        183                HET                                                                                                      
  148         B6N(Cg)                                  22784                                    *Sycp3*^*tm1*.*1(KOMP)Vlcg*^/J           4                  197/214/ 274/279   HOM                                                                                   
  B6N(Cg)     22784                                    *Sycp3*^*tm1*.*1(KOMP)Vlcg*^/J           4                                        165/202/ 274       HOM                                                                                                      
  149         B6N(Cg)                                  23665                                    *Syn3*^*tm1*.*1(KOMP)Vlcg*^/J            2                  267/270            HOM                                                                                   
  B6N(Cg)     23665                                    *Syn3*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        267/270            HOM                                                                                                      
  150         B6.129S1                                 24694                                    *Tgm2*^*tm1Rmgr*^/J                      2                  230/234            HOM                                                                                   
  B6.129S1    24694                                    *Tgm2*^*tm1Rmgr*^/J                      2                                        203/234            HOM                                                                                                      
  151         B6N(Cg)                                  24406                                    *Timm22*^*tm1b(KOMP)Wtsi*^/J             2                  181/192            HET                                                              X                    
  B6N(Cg)     24406                                    *Timm22*^*tm1b(KOMP)Wtsi*^/J             2                                        193                HET                                                                          X                           
  152         B6N(Cg)                                  18639                                    *Timp3*^*tm1*.*1(KOMP)Vlcg*^/J           3                  210/278/ 292       HOM                                                                                   
  B6N(Cg)     18639                                    *Timp3*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        210                HOM                                                                                                      
  153         B6N(Cg)                                  18629                                    *Tmem136*^*tm1*.*1(KOMP)Vlcg*^/J         2                  254/274            HOM                                                                                   
  B6N(Cg)     18629                                    *Tmem136*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        187/274            HOM                                                                                                      
  154         B6N(Cg)                                  23060                                    *Tmem151b*^*tm1b(KOMP)Wtsi*^/J           4                  219/221/ 288       HOM                                                                                   
  B6N(Cg)     23060                                    *Tmem151b*^*tm1b(KOMP)Wtsi*^/J           4                                        202/288            HOM                                                                                                      
  155         B6N(Cg)                                  26542                                    *Tpcn1*^*tm1*.*1(KOMP)Vlcg*^/J           3                  174                HOM                                                                                   
  B6N(Cg)     26542                                    *Tpcn1*^*tm1*.*1(KOMP)Vlcg*^/J           1                                        174                HOM                                                                                                      
  156         B6N(Cg)                                  21752                                    *Tpgs2*^*tm1*.*1(KOMP)Vlcg*^/J           2                  156                HOM                                                                                   
  B6N(Cg)     21752                                    *Tpgs2*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        156                HOM                                                                                                      
  157         B6N(Cg)                                  18606                                    *Tprn*^*tm1*.*1(KOMP)Vlcg*^/J            2                  270                HOM                                                                                   
  B6N(Cg)     18606                                    *Tprn*^*tm1*.*1(KOMP)Vlcg*^/J            2                                        141/293            HOM                                                                                                      
  158         B6N(Cg)                                  18553                                    *Trip13*^*tm1*.*1(KOMP)Vlcg*^/J          4                  181/196/ 239       HOM                                                                                   
  B6N(Cg)     18533                                    *Trip13*^*tm1*.*1(KOMP)Vlcg*^/J          4                                        41/181/321         HOM                                                                                                      
  159         B6N(Cg)                                  21483                                    *Tspan18*^*tm1*.*1(KOMP)Vlcg*^/J         2                  263                HOM                                                                                   
  B6N(Cg)     21483                                    *Tspan18*^*tm1*.*1(KOMP)Vlcg*^/J         2                                        263                HOM                                                                                                      
  160         C57BL/6NJ                                26909                                    *Ttll10*^*em1J*^/J                       2                  200/229            HOM                                                                                   
  C57BL/6NJ   26909                                    *Ttll10*^*em1J*^/J                       2                                        196/229            HOM                                                                                                      
  161         B6N(Cg)                                  22817                                    *Ube2c*^*tm1*.*1(KOMP)Wtsi*^/J           2                  249/270            HET                                                              X                    
  B6N(Cg)     22817                                    *Ube2c*^*tm1*.*1(KOMP)Wtsi*^/J           2                                        287                HET                                                                          X                           
  162         B6N(Cg)                                  24027                                    *Ube2m*^*tm1*.*1(KOMP)Wtsi*^/2J          2                  484                HET                                                              X                    
  B6N(Cg)     24027                                    *Ube2m*^*tm1*.*1(KOMP)Wtsi*^/2J          2                                        484                HET                                                                          X                           
  163         B6N(Cg)                                  23019                                    *Vsig8*^*tm1*.*1(KOMP)Mbp*^/J            1                  116                HOM                                                                                   
  B6N(Cg)     23019                                    *Vsig8*^*tm1*.*1(KOMP)Mbp*^/J            1                                        44                 HOM                                                                                                      
  164         B6N(Cg)                                  23660                                    *Zbtb4*^*tm1*.*1(KOMP)Vlcg*^/J           2                  267                HOM                                                                                   
  B6N(Cg)     23660                                    *Zbtb4*^*tm1*.*1(KOMP)Vlcg*^/J           2                                        144/267            HOM                                                                                                      
  165         B6N(Cg)                                  18620                                    *Zdhhc11*^*tm1*.*1(KOMP)Vlcg*^/J         4                  127                HET                                                                                   
  166         B6N(Cg)                                  24091                                    *Zfp346*^*tm1*.*1(KOMP)Vlcg*^/J          2                  194/202            HET                                                                                   X
  B6N(Cg)     24091                                    *Zfp346*^*tm1*.*1(KOMP)Vlcg*^/J          2                                        194/219            HET                                                                                 X                    
  167         B6N(Cg)                                  23023                                    *Zfp42*^*tm1b(KOMP)Mbp*^/J               2                  151                HOM                                                                                   
  B6N(Cg)     23023                                    *Zfp42*^*tm1b(KOMP)Mbp*^/J               2                                        145                HOM                                                                                                      
  168         B6N(Cg)                                  24057                                    *Zfp961*^*tm1*.*1(KOMP)Mbp*^/J           2                  179/226            HOM                                                                                   
  B6N(Cg)     24057                                    *Zfp961*^*tm1*.*1(KOMP)Mbp*^/J           2                                        179/226            HOM                                                                                                      
  169a        C57BL/6NJ                                22366                                    *Zzef1*^*tm2(KOMP)Vlcg*^/J               2                  198/206            HET                                                                                   
  C57BL/6NJ   22366                                    *Zzef1*^*tm2(KOMP)Vlcg*^/J               2                                        269                HET                                                                                                      
  169b        B6N(Cg)                                  22366                                    *Zzef1*^*tm2*.*1(KOMP)Vlcg*^/J           1                  103                HOM                                                                                   
  B6N(Cg)     22366                                    *Zzef1*^*tm2*.*1(KOMP)Vlcg*^/J           2                                        95/352             HOM                                                                                                      
              Females                                                                                                                                                                                                                                                
              Males                                                                                                                                                                                                                                                  
              Strain not listed as B6N(Cg)                                                                                                                                                                                                                           
  HET         Heterozygous mutant mice                                                                                                                                                                                                                               
  HOM         Homozygous mutant mice                                                                                                                                                                                                                                 
              Normal skin and adnexa                                                                                                                                                                                                                                 
              Lesion in skin or adnexa                                                                                                                                                                                                                               
  X           Prenatal lethal or subviable phenotype                                                                                                                                                                                                                 
              Clinically normal phenotype                                                                                                                                                                                                                            

Nonspecific, sporadic lesions were found in many of the mice in this study. Most commonly these were ruptured hair follicles with hair shaft fragments free in the dermis or hypodermal fat layer surrounded by a variety of inflammatory cell types, usually neutrophils around the hair shaft and a layer of macrophages (trichogranuloma, [Fig 1](#pone.0180682.g001){ref-type="fig"}). These were often solitary and not consistently found in all the mice within a mutant line, which was a primary criterion to differentiate these lesions from gene mutation associated phenotypes. Other mice had follicular dystrophy consistent with C57BL/6 alopecia and dermatitis, a low frequency, strain-specific skin disease resembling human central centrifugal cicatricial alopecia \[[@pone.0180682.ref016]\].

![Incidental lesions were found scattered throughout the skin samples in many lines.\
Incidental lesions were not consistently found in all mice of a line. Lesions included solitary tail skin hair follicle with infundibular plugging (A-C). Truncal hair follicle infundibular plugging, rupture below the level of the sebaceous gland, follicle filled with neutrophils and surrounded by neutrophils and macrophages (D-F). Sporadic skin lesions found in many mice examined included isolated, one or two, ruptured hair follicles in the dermis where the free hair shaft fragments were engulfed by granulomas and surrounded by neutrophils (G-I). Hematoxylin and eosin stain, bar size indicated in each figure.](pone.0180682.g001){#pone.0180682.g001}

Mice with null mutations in their fatty acyl CoA reductase 2 (*Far2*^*tm2b(KOMP)Wtsi*^/2J) gene were found to consistently develop focal alopecia as adults on the dorsal skin extending distally from the base of the ears ([Fig 2](#pone.0180682.g002){ref-type="fig"}). Histologically, in areas where the skin appeared to be relatively normal there were minor lesions affecting the pilosebaceous and Meibomian (eyelids) glands that could be easily over-looked. Other modified sebaceous glands (Zymbal, clitoral, or preputial glands) were not examined in this study. The sebaceous glands superficially appeared to be normal but within the sebaceous duct the sebocytes became brightly eosinophilic and, when these holocrine cells ruptured, their secretion remained present and clumped. By contrast, normal sebocytes were slightly basophilic with fine, uniformly sized vacuoles in their cytoplasm. When the normal cells ruptured there was no evidence of their cytoplasmic content within the follicular infundibulum or on the skin surface ([Fig 3](#pone.0180682.g003){ref-type="fig"}). In areas of alopecia, the epidermis was mildly acanthotic with orthokeratotic hyperkeratosis associated with areas of follicular dystrophy and rupture and an associated foreign body inflammatory response that would lead to follicular scarring, a form of cicatricial alopecia ([Fig 4](#pone.0180682.g004){ref-type="fig"}). Skin surface lipidomic and time course studies are in progress to better define these changes. Further, non-integument phenotypes affecting lipid metabolism, behavior, immunity, and the cardiovascular system have been reported for this strain, and can be found on the IMPC database website (<http://www.mousephenotype.org/data/genes/MGI:2687035#section-associations>).

![A B6N(Cg)-*Far2*^*tm2b(KOMP)Wtsi*^/2J mutant (left) and wildtype (right) mouse.\
Both are 15 week old females. Note the alopecia extending distally from the base of the ears.](pone.0180682.g002){#pone.0180682.g002}

![Sebaceous gland changes in fatty acyl CoA reductase 2 mutant mice.\
Most late stage anagen hair follicles in B6N(Cg)-*Far2*^*tm2b(KOMP)Wtsi*^/2J mutant mice, as in this 184 day old male, were relatively normal and produced a normal hair shaft (A). However, the sebaceous glands were mildly hypoplastic with brightly eosinophilic mature sebocytes that did not immediately rupture as they entered the infundibulum (C). As in other mouse mutations that cause hypoplasia of sebaceous glands, the hair shafts in these mice did not exit the follicular ostium resulting in perforation of the root sheaths, release of the hair shaft into the dermis, and an inflammatory reaction (B). This healed by follicular scarring, or cicatricial alopecia. By contrast, aged and sex matched B6N(Cg) controls rarely exhibited ruptured follicles (D). Sebaceous glands varied in size with the hair cycle, which was normal, and sebocytes had light pink cytoplasm that becomes very pale as the cells matured and abruptly ruptured into the duct. (E). Hematoxylin and eosin stain, bar size indicated in each figure.](pone.0180682.g003){#pone.0180682.g003}

![Follicular dystrophy in fatty acyl CoA reductase 2 mutant mice.\
In areas of alopecia, the B6N(Cg)- *Far2*^*tm2b(KOMP)Wtsi*^/2J mutant skin was mildly acanthotic and orthokeratotic, most likely secondary to the abnormal secretions of the sebaceous glands. The infundibulum was mildly dilated (A, B). Follicles with follicular dystrophy (C, D) and with pigment cast (E) were identified occasionally to frequently. Such ruptured follicles with granulomatous inflammation eventually lead to follicular scarring, a form of cicatricial alopecia. Hematoxylin and eosin stain, bar size indicated in each figure.](pone.0180682.g004){#pone.0180682.g004}

Heterozygous mutant mice for the protein phosphatase 1, regulatory subunit 9B (*Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J) gene had between one and five vibrissae hair shafts undergoing degenerative changes (follicular dystrophy) in each of three females and two males examined. Each section contained 10--15 vibrissae follicles ([Fig 5](#pone.0180682.g005){ref-type="fig"}). Only mystacial vibrissae were affected, not the periorbital or lower leg vibrissae. No other hair types were affected. This change was not found in two female and two male homozygous mutants suggesting that this was a nonspecific background lesion, as vibrissae follicular dystrophy was also seen occasionally in many mouse lines but only in one or two follicles and not consistently in all vibrissae in any section.

![Vibrissae dystrophy in some protein phosphatase 1, regulatory subunit 9B mice.\
B6N(Cg)-*Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J, had follicular dystrophy limited to many but not all mystacial vibrissae in heterozygous but not homozygous mutant mice suggesting this is an incidental, albeit relatively frequent lesion. Boxed areas are enlarged as indicated by the arrows. Pigment casts are identified in all dystrophic hair follicles (B, F, and H). Hematoxylin and eosin stain, bar size indicated in each figure.](pone.0180682.g005){#pone.0180682.g005}

The skin and nail sections from almost all of the mice in these studies were clinically and histologically normal.

Discussion {#sec003}
==========

We have investigated the apparent deficiency of skin and adnexal phenotypes in a random set of strains from a systematic, genome-wide, knockout study using histopathological phenotyping. We report here that numbers detected confirm an earlier study \[[@pone.0180682.ref014]\] which also showed low numbers of detectable skin phenotypes, suggesting that ascertainment is not the cause of the "missing phenotypes".

Of over 100 homozygous mutants screened, only one line, *Far2*^*tm2b(KOMP)Wtsi*^/2J, had lesions affecting sebaceous glands with what appeared to be a secondary follicular dystrophy, which probably progresses to a form of cicatricial alopecia as seen with a number of other mouse mutations with abnormalities of the sebaceous glands \[[@pone.0180682.ref017]\]. A rare autosomal recessive neurological disease without hair abnormalities was reported for humans with mutations in *FAR1* \[[@pone.0180682.ref018]\]. While the biochemistry of these two proteins, FAR1 and FAR2, is known no disease is currently associated with mutations in *FAR2* in humans (<http://omim.org/entry/616156?search=Far2&highlight=far2>). However, *Far2* transcripts were reported in the eyelid and skin, presumably localized in the Meibomian and sebaceous glands, respectively \[[@pone.0180682.ref019]\]. Information obtained from phenotyping these mice will be useful to help assess sebaceous gland abnormalities in humans as not all genes that are expressed in sebaceous glands result in sebaceous gland abnormalities or clinically obvious hair problems, such as mouse null mutations in *Soat1* \[[@pone.0180682.ref020], [@pone.0180682.ref021]\]. Detailed studies are in progress to define that pathogenesis of the follicular dystrophy and to match it with a specific human disease in these *Far2* mutant mice.

A second line, *Ppp1r9b*^*tm1*.*1(KOMP)Vlcg*^/J, had follicular dystrophy limited to many but not all vibrissae in heterozygous but not homozygous mutant mice suggesting this was a nonspecific background problem. A targeted mutant mouse line made for this gene (*Ppp1r9b*^*tm1Jfe*^) was reported to only have neurological abnormalities \[[@pone.0180682.ref022], [@pone.0180682.ref023]\]. As the vibrissae are an important somatosensory organ in the mouse \[[@pone.0180682.ref024]\] this structure should be carefully evaluated in such phenotyping studies.

The International Mouse Phenotyping Consortium has provided a unique, internally consistent dataset with genome-wide coverage and has yielded a great deal of valuable information on gene function. At the outset anticipation was high that systematic creation of null mice for all known protein coding genes would yield large amounts of information on the pathobiology of human disease. The high frequency of embryonic and neonatal lethal lines \[[@pone.0180682.ref004]\], consistent with previous estimates \[[@pone.0180682.ref006]\], was not a surprise, but potentially limits the value of these mice for detailed investigation of adult phenotypes and diseases which will require carefully designed conditional strategies.

The relatively low frequency of skin phenotypes in the International Mouse Phenotyping Consortium mice found in this and an earlier study \[[@pone.0180682.ref011]\] was surprising given the frequency expected from global surveys of MGI and OMIM discussed above. The histopathological survey presented here was very detailed focusing on the skin, and we believe this supports the assertion that the "missing phenotypes" are not due to lack of ascertainment, at least in the random sample selected. The remaining possibilities are that genes involved in skin morphogenesis and physiology are overrepresented in those essential for embryogenesis, the C57BL6N background is failing to show phenotypes, or that the complete removal of gene function in the mouse knockouts have more severe functional consequences than the point mutations, regulatory lesions, and insertions/deletions (indels) that constitute most disease-causing mutations in humans. Protein truncating variants which provide the greatest likelihood of complete functional loss ("knockouts") are very rare in the human population \[[@pone.0180682.ref025]\].

In the current study, 74% of the heterozygous lines were pre-wean lethal or sub-viable as homozygotes (that were not examined) presenting the possibility that mutants having skin phenotypes might also be embryonic essential genes. While it is possible to argue that essential genes should never show up as disease genes in a population due to negative selection, there is increasing evidence that essential genes are often 'disease genes"--i.e. carry mutations that are associated with abnormal or disease phenotypes. This is by no means a settled conclusion however. The relationship between gene essentiality and disease association is discussed in detail in Cooper et al. \[[@pone.0180682.ref025]\].

Studies conducted around a decade ago, based on a limited gene set, suggested that mouse genes that are essential---i.e., genes related to more severe phenotypes in mouse, are more likely to be human disease genes, but that embryonic lethals were less likely \[[@pone.0180682.ref026]\]. However, a more recent study strongly suggested that about 30% of human disease genes (from OMIM) have a mouse ortholog with a lethal knockout phenotype (673/1965) \[[@pone.0180682.ref027]\]. The discrepancies here reflect to an extent the methodology used to calculate the association and the inclusion or exclusion of genes with no phenotypic data in the study by Park *et al*. \[[@pone.0180682.ref026]\]. This association will certainly stand further scrutiny now that more data are available, but does not explain why the IMPC/KOMP strains show fewer skin phenotypes than the bulk of the loss-of-function allele carrying strains in MGI. Core analysis carried out on Ingenuity^™^ using the lists of lethal essential genes from supplementary table 3 of Dickinson *et al*. \[[@pone.0180682.ref004]\] shows no significant enrichment of annotations to dermatological disease or skin development, mitigating against the possibility that genes associated with skin disease are overrepresented in the essential segment of the genome. A working conclusion, therefore, is that either there is a problem with the strain background in this resource showing full expressivity of mutations affecting the skin, or that we have so far insufficient coverage of the genome using high-resolution phenotyping to account for the "missing phenotypes" from the International Mouse Phenotyping Consortium program. Of concern is the observation that most human disease associated mutations are hypomorphic alleles which, in the event of lethal homozygosity of complete KOs, are much more likely to present a phenotype compatible with survival. The potential for missing these with a complete loss of function strategy is significant, especially when examining completely novel genes where there are no existing experimental or inferred annotations. The power of looking for skin phenotypes using a strategy such as that developed by Beutler *et al*. \[[@pone.0180682.ref028]\] using ENU mutagenesis is very persuasive. Currently Mutagenetix (<https://mutagenetix.utsouthwestern.edu/home.cfm>), accessed 1 Mar 2017) shows 49 genes with skin associated phenotypes recovered as phenotypic mutations from a total of 262 phenotyped genes (119 of 498 mapped mutations) and should be considered in the future along with full characterization of spontaneous and ENU generated mutations in large collections.

Materials and methods {#sec004}
=====================

Generation of mutant mice {#sec005}
-------------------------

### Embryonic stem cell lines {#sec006}

Initially mice included in the program were produced from embryonic stem cell lines acquired from repositories (<https://www.komp.org/> or <http://www.eummcr.org/>). These resources comprised two major allele types, the Velocigene "definitive null" allele produced by the Regeneron Corporation (*Vlcg*) \[[@pone.0180682.ref001]\] and the "knockout first, conditional ready" design produced by the Wellcome Trust Sanger Centre and partners (Sanger) \[[@pone.0180682.ref002]\]. The *Vlcg* alleles delete the entire coding sequence of the target gene, including all introns, while inserting a *LacZ* reporter cassette. The *neo* resistance cassette is flanked by *loxP* sites for removal by *cre*-recombinase. The Sanger allele is a multifunctional design. In its native form, the strong splice acceptor typically generates a null allele. When bred to *Flp*-expressing mice, the entire 5' *LacZ* and *neo* cassette are removed leaving a *loxP*-flanked critical exon for conditional mutagenesis. When bred to *cre*-recombinase transgenic mice, this allele is converted into a "definitive null" which retains the 5' *LacZ* cassette and removes the critical exon and *neo* marker.

All animal work was approved by The Jackson Laboratory Animal Care and Use Committee (approval number 07005 \"Mouse Models for Dermatology Research").

### Generating live mice {#sec007}

Following importation at The Jackson Laboratory, clones are injected into blastocyst hosts and resulting chimeras are bred to C57BL/6NJ (STOCK\#5304) or C57BL/6NJ-*Tyr*^*em3J*^/GrsrJ (STOCK\#21999; to select for coat color transmission) inbred mice (see [Table 1](#pone.0180682.t001){ref-type="table"}) to assure isogenicity of the resource. Germline transmitting (GLT) clones are quality confirmed (both *Vlcg* and Sanger) for targeting. Following GLT, mice are bred to congenic *cre*-recombinase transgenic mice (B6N.Cg-Tg(*Sox2-cre*)1Amc/J; STOCK\#14094) to remove the *neo* cassette and to generate a definitive null allele, generating the tm1.1 allele (*Vlcg* and non-conditional Sanger clones) and *tm1b* allele (from conditional-ready Sanger clones). Four lines were generated by CRISPR/Cas9 mutagenesis, using standard approaches \[[@pone.0180682.ref029]\] to generate small deletions in the early part of the transcript. These mice are expanded, quality control-confirmed, and intercrossed to generate knockout mice. Approximately 30% of lines display lethality or sub-lethality (defined as yielding less than 12.5% homozygous mice) \[[@pone.0180682.ref004]\]. For these strains, heterozygous mice were phenotyped. For other strains, homozygous breeder pairs established for colony maintenance used in this study when they became retired breeders to maximize chances of finding adult onset diseases.

Systematic analysis of mutant mice {#sec008}
----------------------------------

### Animal logistics {#sec009}

Two female and two male retired breeder mice were provided from The Jackson Laboratory knockout mouse production program ([Table 1](#pone.0180682.t001){ref-type="table"}). Homozygous lethal embryos and perinatal lethal mouse lines were specifically excluded from these studies. In these cases, heterozygous animals were aged and evaluated. All lines, whether *Vlcg* definitive null or Sanger conditional-ready mice were evaluated post-*cre* excision, which removed the *neo* cassette and heterologous promoter and in the case of Sanger alleles also removing the critical exon to create a definitive null allele (*Vlcg tm1*.*1* and Sanger *tm1b*). All mice had skin, hair, and nails collected (see below).

### Necropsy procedures, slide generation, and histopathologic analyses {#sec010}

As the skin is anatomically and molecularly distinct throughout the body in both mice and humans \[[@pone.0180682.ref030], [@pone.0180682.ref031]\] and diseases can be restricted to specific anatomic sites, skin was collected from several areas. As dorsal and ventral trunk skin are histologically similar in the mouse, they were collected with other anatomically identifiable tissues and put into respective cassettes: 1) dorsal skin and ear skin; 2) ventral skin, eyelid skin, muzzle skin; 3) digits to include the foot pads and nail unit; and 4) cross and longitudinal sections of the tail. This allows for evaluation of all major hair follicle types, mucocutaneous junctions, the nail unit, as well as the footpad (including eccrine glands) \[[@pone.0180682.ref032]\]. Detailed protocols on skin removal, orientation, and processing are described elsewhere \[[@pone.0180682.ref033]\]. Tissues were fixed in Fekete's acid-alcohol-formalin solution. Sections were stained with hematoxylin and eosin (H&E) for routine screening.

Far2

:   fatty acyl CoA reductase 2 gene

GLT

:   germ line transmitting

H&E

:   hematoxylin and eosin stain

MGI

:   Mouse Genome Informatics Database

[^1]: **Competing Interests:**JPS, KAS, and VEK have a research contract with BIOCON LLC which has no relevance to this project. The remaining authors (SSD, SAM, BAS, PNS, and CHP) have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
