The Gtf2i duplication allele (Gtf2iDup; Dp(5Gtf2i-Gtf2ird1)1Lro) was created by Dr. Lucy R. Osborne (University of Toronto) by first obtaining two independent mouse lines with loxP sites at the desired endpoints, and then using Cre recombinase expression during male meiosis to generate a chromosome with one additional copy of a functional Gtf2i gene. Specific details are below.
The Gtf2i3'UTRloxP allele (Gtf2itm1Lro) was created by Dr. Osborne using a 5' hprt vector to place a loxP site and neo cassette into the 3' UTR of the general transcription factor II I locus (Gtf2i) on chromosome 5. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+ derived R1 embryonic stem (ES) cells. Chimeric males were bred with CD1 females to establish the colony. The Gtf2i3'UTRloxP mice were bred at least ten generations with CD1, and then used as described below.
The XS0608 allele (Gtf2ird1Gt(XS0608)Wtsi) has a loxP site inserted into intron 4 of the general transcription factor II I repeat domain-containing 1 locus (Gtf2ird1) on chromosome 5. First, the pGT0lxf gene trap vector was obtained containing (from 5' to 3') a mouse En2 intron 1, a lox71 site, a mouse En2 exon 2 splice acceptor, a loxp site, a β-geo fusion reporter protein (β-galactosidase and neomycin phosphotransferase), an SV40 polyadenylation sequence and a frt site. The vector was electroporated into 129P2/OlaHsd-derived E14TG2a.4 ES cells. ES cells with the gene trap vector randomly inserted into Gtf2ird1 intron 4 were identified and named line XS0608. Chimeric males were bred with CD1 females to establish the colony. The XS0608 mice were bred at least five generations with CD1, and then used as described below.
To generate the Gtf2iDup allele (Dp(5Gtf2i-Gtf2ird1)1Lro), Dr. Osborne first bred CD1.Gtf2i3'UTRloxP animals to mice expressing Cre recombinase during male meiosis (Sycp1-Cre transgenic mice bred more than ten generations on CD1; see Stock No. 003466). The CD1.Gtf2i3'UTRloxP::Sycp1-Cre double mutant was then bred to CD1.XS0608 mice; producing the triple mutant (CD1.Gtf2i3'UTRloxP::XS0608::Sycp1-Cre). Subsequent male germline recombination of the loxP sites produces some male gametes with a chromosome 5 composed of the centromeric sequences from the XS0608 allele, a single remaining loxP site, and then the telomeric sequences from the Gtf2i3'UTRloxP allele; i.e., duplication of a functional Gtf2i gene and a non-functional Gtf2ird1 allele (see additional note below). Breeding to wildtype CD1 outbred females transmitted the Gtf2iDup allele to offspring.
Gtf2iDup mice were backcrossed nine generations with C57BL/6J inbred mice (the Cre transgene was removed and the Y chromosome was made C57BL/6J), and then sent to The Jackson Laboratory Repository in 2015. Upon arrival, males were used to cryopreserve sperm. To generate our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Additional note: the Gtf2i3'UTRloxP-derived sequences that remain on the duplication region do not include the neo cassette. In addition, the XS0608-derived Gtf2ird1 sequences that remain on the duplication region (including the loxP::β-geo fusion::SV40pA::frt and downstream Gtf2ird1 coding exons) result in a null allele because the upstream Gtf2ird1 sequences and gene trap splice acceptor sequences are absent.
