Model Systems for Studying Kisspeptin Signalling: Mice and Cells - Kisspeptin Signaling in Reproductive Biology - page 477

of the Kiss1 gene, with prominent expression in the ARC and AVPV regions and
appropriate regulation by estradiol; although some expression has also been found in
cortical neurons (see below). Surprisingly, homozygous mutant mice of both sexes
do not show severe fertility defects. The mutant males appear to be nearly normal,
with small testes, but they can impregnate females and have normal testosterone
levels, whereas the females have markedly impaired fertility, but relatively normal
organ weights (Robert Steiner, personal communication). In situ hybridization
showed a reduction in the level of Kiss1 transcripts, so this transgenic mouse model
probably carries a hypomorphic mutation rather than a null allele. It is noteworthy
that there is a moderately conserved splice acceptor sequence (AGGAGACTGTAGAC)
located 21 bases upstream from the ATG codon which may allow some transcripts to
splice out the Cre / Gfp transgene and allow low levels of kisspeptin protein to be
made. Since kisspeptins are very potent stimulators of GnRH secretion, low levels
may be suffi cient to maintain the reproductive axis.
The Kiss1 tm1.1 ( cre ) Uboe mice have an IRES-Cre transgene inserted just after the
Kiss1 coding region in exon 2 (Fig. 22.1 ) [ 43 ]. Appropriate expression of the
Cre transgene in ARC and AVPV Kiss1 neurons was confi rmed by breeding
the mice with a line expressing a CRE-activated fl uorescent reporter and 97% of
Kiss1 neurons were fl uorescently labelled.
In addition to the various transgenic mouse lines that have been generated by
gene targeting, several Kiss-Cre lines have also been made by pronuclear microin-
jection of bacterial artifi cial chromosome (BAC) constructs. One of these BAC vec-
tors contained a DNA fragment with 109 kb of genomic sequence upstream of a Cre
transgene inserted at the Kiss1 translational initiation codon [ 44 ]. Three founder
lines were generated (J2-3, J2-4 and J2-6), and to validate the expression profi le of
these lines, the mice were bred with CRE-dependent reporter mice (GFP or
β -galactosidase). The Tg ( Kiss1 - Cre ) J2 - 4Cfe line showed the most appropriate

expression of the Cre transgene with GFP or -galactosidase expression being

found in the AVPV and the ARC regions of the hypothalamus and 93% of these
neurons were identifi ed as co-expressing Kiss1 mRNA by in situ hybridization. The
Tg ( Kiss1 - Cre ) J2 - 3Cfe line showed expression in the ARC, but had very little
expression in the AVPV region. The reason for the more limited expression pattern
found in the Tg ( Kiss1 - Cre ) J2 - 3Cfe mice is not known, but probably results from
rearrangement of the Kiss1 promoter during the BAC integration event and loss of
important regulatory elements. Expression of the Tg ( Kiss1 - Cre ) J2 - 4Cfe transgene
was also found in the cerebral cortex, which is not a site where Kiss1 expression has
been found in adult mice. This ectopic expression could arise from inappropriate
Kiss1 promoter activity as a result of the site of the transgene integration.
Alternatively, the expression in the cortical neurons could indicate that these cells
expressed Kiss1 at some point in their development while in the adult this expres-
sion is no longer found. Since the Kiss1 -Cre transgene was visualized by CRE-
mediated activation of a reporter gene, any cell that even transiently expressed Kiss1
will be permanently marked. This phenomenon has also been observed in the
Kiss1 tm1.1 ( cre / EGFP ) Stei mice, when expression was visualized using a CRE-inducible
LacZ gene and

β

β -galactosidase positive neurons were found extending outside