Model Systems for Studying Kisspeptin Signalling: Mice and Cells - Kisspeptin Signaling in Reproductive Biology - page 471

environment and the data extrapolated to other species, including humans.
Manipulation of embryonic stem (ES) cells allows precise genetic modifi cations to
be introduced into mice [ 24 ]. These modifi cations range from precise genetic altera-
tions such as deletions, point mutations and reporter/modulator gene tagging to
more extensive mutations such as conditional alleles, chromosomal deletions and
translocations [ 25 ].
Transgenic Mice with Global Disruption of Kiss1r
Five transgenic mouse lines have been generated with global disruption of the
Kiss1r gene (Fig. 22.1 and Table 22.1 ). The Kiss1r tm1Coll line has a 702 bp deletion
encompassing the fi nal 92 bp of exon 1, the whole of intron 1 (509 bp) and the fi rst
101 bp of exon 2 [ 26 ]. The Kiss1r tm1Gstn line has a 52 bp deletion within exon 2 [ 27 ],
while the Kiss1r tm1.1On line has a deletion of all fi ve coding exons after a CRE-
mediated recombination event [ 28 ]. These three transgenic lines all contain a LacZ
reporter gene driven from the Kiss1r promoter and can be used to map the expres-
sion pattern of the kisspeptin receptor in the mouse brain [ 9 ]. The Kiss1r tm1Rla line
has complete deletion of exon 2 [ 29 ], while the Kiss1r Gt1Stei line has a proviral inser-
tion in intron 2 with no loss of Kiss1r coding sequence [ 30 , 31 ]. Kiss1r expression
is disrupted by a splice acceptor within the retrovirus to trap splicing from exon 2
and two polyadenylation signal sequences to terminate transcription. In situ hybrid-
ization could not detect any Kiss1r transcripts in the mutant mice, but as the coding
exons are still intact it is at least theoretically possible that there might be a very low
level of residual expression (i.e. a hypomorphic mutation). The retrovirus also
encodes the reverse tetracycline-dependent transactivator protein (rtTA), which
should be expressed in cells where the Kiss1r gene is transcriptionally active. The
Gfp gene in the provirus will, however, not specifi cally mark neurons expressing the
kisspeptin receptor, as it is driven by its own promoter. In the absence of an antibody
with good specifi city for the kisspeptin receptor, confi rmation that these mice have
null alleles has relied on molecular means such as RT-PCR or in situ hybridization.
Transgenic Mice with Global Disruption of Kiss1
Three lines of transgenic mice have been described with global disruption of
the Kiss1 gene. In the Kiss1 tm1Coll line the whole of the Kiss1 coding region has
been deleted and replaced with an IRES LacZ sequence [ 32 ]. Similarly, in the
Kis1 tm2 ( cre / EGFP ) Coll line, the coding exons have been replaced with a Cre / Egfp fusion
gene from the pCAG-Cre:GFP vector from Connie Cepko's laboratory [ 33 , WHC,
unpublished]. In the Kiss1 tm1Rla line, the fi rst Kiss1 coding exon that contains the
translational initiation codon has been deleted [ 29 ]. Absence of kisspeptin expres-
sion in these mutant mice has been confi rmed by RT-PCR and by immunohisto-
chemistry using a well-characterized and specifi c anti-kisspeptin antibody [ 34 ].