  Cellular barcoding is a significant, recently developed, biotechnology tool
that enables the familial identification of progeny of individual cells in
vivo. Most existing approaches rely on ex vivo viral transduction of cells with
barcodes, followed by adoptive transfer into an animal, which works well for
some systems, but precludes barcoding cells in their native environment, such
as those inside solid tissues. With a view to overcoming this limitation, we
propose a new design for a genetic barcoding construct based on the Cre Lox
system that induces randomly created stable barcodes in cells in situ by
exploiting inherent sequence distance constraints during site-specific
recombination. Leveraging this previously unused feature, we identify the
cassette with maximal code diversity. This proves to be orders of magnitude
higher than what is attainable with previously considered Cre Lox barcoding
approaches and is well suited for its intended applications as it exceeds the
number of lymphocytes or hematopoietic progenitor cells in mice. Moreover, it
can be built using established technology.
