Biology Reference
In-Depth Information
23.2.3.2
Embryo generation, detection of BAC transgenic offspring,
and breeding strategy
Once the BAC constructs containing mutant LHCGRs were generated, they required
preparation for pronuclear injection. The BAC constructs were linearized and recon-
stituted in microinjection buffer (100 mM EDTA, 10 mM Tris-HCL pH 7.5, with
additional 30 mM spermine and 70 mM spermidine to aid integration of the entire
BAC construct into the genome) by buffer exchange. The BAC were injected into
fertilized mouse oocytes (our mouse background of choice was FVB/N) and
implanted into surrogate FVB/N mothers using standard methodology, as previously
described (
Rivero-M
ยจ
ller et al., 2010
).
To determine the genotype of the resulting litter, we took an earlobe sample from
each animal at day 15 postpartum. The genotype was determined using standard
DNA extraction and PCR protocols using primers that distinguished between the
WT
Lhcgr
,
Lhcgr
B
, and
Lhcgr
S
. Primer design is listed in
Table 23.1
. Mice car-
rying either
Lhcgr
B
or
Lhcgr
S
were retained and used for subsequent matings to
obtain the homozygous
LuRKO
/
Lhcgr
B
/
Lhcgr
S
genotype for studying LHCGR
transactivation. Since the LuRKO mice are infertile, heterozygote animals were re-
quired for breeding. The breeding strategy required two distinct stages as detailed in
Fig. 23.3
.
23.2.3.3
Analysis of male reproductive tract and testes
Assessment of LHCGR transactivation on downstream cellular signaling events is
ultimately determined by the degree of functional rescue of the infertile and hypo-
gonadal reproductive phenotype of the LuRKO animals. To date, our studies have
focused on the male LuRKO animals. In males, the primary function of the LHCGR
expressed in the Leydig cells of the testes is production of testosterone (
Fig. 23.4
(I)).
The downstream actions of LHCGR are to maintain steroidogenesis through regulat-
ing expression of key Leydig cell-specific steroidogenic genes, such as
StAR
and
CYP17a1
, to drive testosterone production and facilitate spermatogenesis
(
Fig. 23.4
(I), reviewed by
Huhtaniemi & Alevizaki, 2007
). Therefore, the best de-
terminates of functional rescue of the LuRKO animals by LHCGR transactivation
Primer Design for Genotyping of LHCGR
B
, LHCGR
S
, and LuRKO
Table 23.1
Animals
Primer ID
Sequence
5
0
-GTATTCAACAAGGGGCTGAAGG-3
0
IRES forward
eCFP (LHCGR
B
) reverse
5
0
-TTGATCCTAGCAGAAGCACAGG-3
0
RFP (LHCGR
S
) reverse
5
0
-CCATGGTCTTCTTCTGCATCAC-3
0
5
0
-TCTGGGGATCTTGGAAATGA-3
0
WT LHCGR forward
5
0
-CACCTTGACACCTGGAGT-3
0
WT LHCGR reverse
5
0
-GGGCTCTATGGCTTCTGAGGCGGA-3
0
LuRKO (neo) forward
5
0
-TCTCAGGGAGGATTTGGGTATGG-3
0
LuRKO (neo) reverse
