Abstract
Pax6 is a member of a highly evolutionarily conserved family of transcription
factors. These genes are characterised by the presence of a 'paired-type' DNA
binding domain. Pax6 is developmentally regulated and is required for the normal
embryonic development of the central nervous system, eye and pancreas. In adults it
is thought to be involved in the correct function of the pancreas and cerebellum,
although its precise mechanism of action is not as yet fully understood.
In order to better understand Pax6 function I generated a novel tool - a 'Pax6
reporter' transgenic mouse that expresses GFP under the control of Pax6 regulatory
elements. The transgenic mouse was generated from a modified yeast artificial
chromosome (YAC) that contains the human PAX6 gene and has been previously
demonstrated to rescue loss of endogenous Pax6 in Pax6seysey mice.
The key advantages of a YAC addition transgenic include that it is already known
that Pax6 regulatory elements are present over a 200Kb region and inserting a
reporter gene into the endogenous Pax6 would not be independent of the endogenous
locus.
An expression cassette encoding GFP and an IRES-neoR vector were inserted into
the YAC in frame with the normal PAX6 translation start point in exon 4. preserving
the rest of the PAX6 locus. This put GFP and neomycin under the control of the
PAX6 regulatory elements. The modified YAC was then injected into fertilised
mouse oocytes to generate nine lines of transgenic mice.
Once generated the expression pattern in each line was analysed at a range of
developmental stages by imaging appropriate sections of agarose embedded mouse
embryos. This confirmed that the expression was the same as the previously reported
Pax6 expression pattern. In addition, the copy number and extent of the YAC
incorporated in each of the nine lines was investigated.
