Plasmid pGS116 contains the human cytomegalovirus (hCMV) promoter,
followed by a Simian virus 40 (SV40) intron, the human alpha-1
antitrypsin (hAAT) cDNA and the SV40 polyadenylation site. Plasmid
pGS119 was used to transform primary human amniocytes and contains the
E1 and pIX region of adenovirus type 5 (Ad5) from nt. 505 to 4079. E1A
is under the control of the murine phosphoglycerate kinase (pgk)
promoter, while E1B and pIX expression is controlled from their natural
promoters. The E1B downstream intron, splice acceptor and polyA signal
were replaced by corresponding motifs from SV40.
Primary cells and cell lines
Primary amniocytes were derived from routine clinical amniocentesis
usually performed during week 16–20 of gestation for prenatal
diagnosis. Amniocytes were obtained including patients informed consent
as redundant material not required for clinical diagnosis. Thus, the
amniotic fluid was used in accordance with all legal and ethical
requirements. Primary cells were cultivated in Ham's F10 medium
supplemented with L-glutamine (Invitrogen), 10% fetal calf serum (FCS),
2% Ultroser G (BioSpepra) antibiotic/antimycotic (Invitrogen) in 5% CO2 at
37°C. After several passages cells were stepwise adapted to Opti-Pro
medium (Invitrogen), supplemented with Ultroser. HEK-293 cells were
cultivated in alpha modified Eagle's medium (αMEM, Invitrogen) supplemented with 10% FCS and antibiotics in 5% CO2 at 37°C.
Transfection and expansion of transformed amniotic cells
Five 6-cm dishes with subconfluent cultures of amniocytes adapted to Opti-Pro medium (see above) were transfected with 1 μg
per dish of each pGS116 and pGS119 using Effectene transfection kit
(Qiagen). Both plasmids were digested with ScaI prior to transfection.
The next day, transfected cells were transferred to 15-cm dishes and
Ultroser was reduced to 1%. About 20 to 30 days post transfection cells
from each dish were transferred to two 15-cm dishes; individual cell
clones arising from single transformed cells appear on the transfected
dishes several days later. These clones were expanded as clone pools by
passaging the cells on individual dishes and further cultivation for
several passages until untransformed amniocytes stopped growing and
were overgrown by transformed cells. Genetically identical clones of
two pools of Z171 in passage 26 (for Z171-5A) and 27 (for Z171-5B) were
isolated by limited dilution in 96-well plates and further expanded.
Passage numbering of cloned cells started again with passage one in
Expression of E1 proteins
For detection of E1 protein expression Western Blots were performed using monoclonal antibodies. 7 × 105 cells/well
from Z171 cell pools were plated on 6-well plates and harvested 72 h
later by detaching the cells in Tris-Saline/4 mM EDTA, pelleting and
lysing cells in loading buffer containing SDS. For control either
untransfected primary amniocytes or untransfected HEK293 cells were
used. The proteins were separated in a 10% SDS-polyacrylamid gel,
transferred to nitrocellulose and incubated with either an anti-E1A or
an anti-E1B-21kD antibody (Oncogene Research), and anti-mouse (E1A,
Jackson ImmunoResearch Laboratories) or anti-rat (E1B-21kD, Oncogene
Research) as secondary antibody and visualized by chemoluminescence.
Expression of hAAT (Western Blot)
The intracellular and secreted hAAT expression in different cell
lines were analysed in a Western Blot using a monoclonal antibody. 7 ×
105 cells/well from each Z171 cell pool were plated on
6-well plates. Seventy-two hours later the supernatants were collected,
cells were counted and lysed as described above. For detection of
intracellular expression of hAAT proteins, cell lysates were separated
on a 10% SDS-Gel. For detection of secreted hAAT, 10 μl
of the cell culture media were applied on a 10% SDS-gel. Intracellular
and secreted hAAT was visualized by chemoluminescence using a
monoclonal anti-hAAT antibody (ICN Biomedicals), and an anti-goat
antibody (Pierce). As negative controls, proteins from untransfected
primary amniocytes were used. As positive control, hAAT purified from
human plasma (ICN Biomedicals) was used.
Quantitation of hAAT expression (ELISA)
The amount of hAAT secreted into the cell culture medium was
quantitated using the enzyme-linked immunoabsorbent assay (ELISA). From
different passages of each Z171 cell pool 7 × 105 cells/well
were plated on 6-wells plates. Forty-eight or 72 hours later the
supernatants of the different cell lines were collected. The amount of
hAAT secreted into the medium was quantitated by ELISA using polyclonal
hAAT-antibodies (HRP-coupled and uncoupled, ICN Biomedicals). As
standard, different amounts of hAAT purified from human plasma were
used. The hAAT levels were either calculated in μg expressed per ml medium or as pg/cell expressed in 24 h per cell using the numbers of cells initially plated.
Glycoanalyses of hAAT
The glycosylation of hAAT expressed in Z171-5A and Z171-5B was
analysed by cleaving N-linked oligosaccharides from hAAT using the
peptide-N-glycosidase F (PNGaseF). To 22.5 μl cell culture medium, 2.5 μl of denaturing buffer (5% SDS, 10% β-Mercaptoethanol) was added. After denaturing for 10 min at 96°C, 3.5 μl G7-reaction buffer (New England Biolabs, 10× containing 500 mM Na-phosphate buffer), 3.5 μl 10% NP-40 and different amounts of PNGaseF (500, 50, 5 and 0 U/μl, New England Biolabs) were added and incubated for 1 h at 37°C. For control either hAAT purified from human plasma or 22.5 μl of cell culture medium were denatured and incubated either with PNGaseF or H2O as described above.
For detection of sialic acid linked to galactose residues, hAAT
secreted into the cell culture medium was incubated with Neuraminidase.
This enzyme catalyzes the hydrolysis of α2–3, α2–6 and α2–8 linked N-acetyl-neuraminic acid residues from glycoproteins. To 22.5 μl cell culture or medium plasma derived hAAT, 2.5 μl G1-reaction buffer (New England Biolabs, 10× containing 500 mM sodium citrate) and 3 μl Neuraminidase (50 U/μl, New England Biolabs) or 3 μl H2O was added and incubated for 1 h at 37°C.
Controls and digested proteins were separated in an
SDS-polyacrylamide gel and Western Blotting using anti-hAAT antibodies
was performed as described above.