B6.129(Cg)-Dp(5Gtf2i-Gtf2ird1)1Lro/JcrwJ

Stock number: 027792
Product name: Gtf2i duplication (Gtf2i^Dup)
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research

Description

The Gtf2i duplication allele (Gtf2i^Dup) has one additional copy of a functional mouse general transcription factor 2I gene (TFII-I). These mice model characteristics of 7q11.23 duplication syndrome (Dup7q11.23), and may be useful for studying the cellular mechanisms underlying GTF2I-related neurodevelopmental disorders and molecular separation-anxiety pathways.

Detailed description

The Gtf2i duplication allele (Gtf2i^Dup; Dp(5Gtf2i-Gtf2ird1)1Lro) has one additional copy of a functional mouse Gtf2i gene (and a non-functional Gtf2ird1 allele) inserted centromeric to the endogenous general transcription factor 2I locus (GTF2I; encoding protein TFII-I).

TFII-I is a transcriptional activator that regulates neuronal maturation and intracellular Ca2+ levels (through the TRPC3 channel), and is highly expressed in the prenatal and postnatal developing brain. The human chromosome 7 locus, 7q11.23, is a 1.5-1.8 Mbp region susceptible to genomic rearrangement through non-allelic homologous recombination. This region contains ~25 protein coding genes; including GTF2I. 7q11.23 duplication syndrome (Dup7q11.23) and 7q11.23 deletion syndrome (Williams-Beuren Syndrome [WBS]) are neurodevelopmental disorders with unique/contrasting cognitive and behavioral profiles. In humans, Dup7q11.23 is associated with speech and language delay, anxiety (both social and nonsocial) and autism. Some individuals exhibit macrocephaly and increased brain volume, as well as cortical dysplasia. Approximately 30% of 4- to 12-year-olds with Dup7q11.23 may meet diagnostic criteria for a separation-anxiety disorder.

Mice that are wildtype (Gtf2i^+/+), hemizygous (Gtf2i^Dup/+) and homozygous (Gtf2i^Dup/Dup) for the Gtf2i duplication allele harbor a total of two, three and four Gtf2i copies, respectively. When compared to mouse pups with one Gtf2i copy (heterozygous for a knockout allele) or two Gtf2i copies (wildtype), pups with additional Gtf2i copies (hemizygous and homozygous mice) show significantly increased maternal separation-induced anxiety as measured by ultrasonic vocalizations. Copy number alterations of GTF2I affect cortical neuronal morphology/maturation, novel object recognition and agonist-induced calcium entry (calcium signaling via the TRPC3 channel). Specifically, Gtf2i^Dup/+ mice exhibit deficits in a cortically-dependent learning and memory task (novel object recognition). Ca2+ influx via neuronal TRPC3 channels is decreased in Gtf2i^Dup/+ (whereas it is increased in mice heterozygous for a knockout allele).

The donating investigator reports hemizygous and homozygous mice are viable and fertile with healthy nutritional status. When maintaining their colony by breeding wildtype (noncarrier) females with hemizygous males, they report the mice are good breeders with no reduced survival. They also report no GTF2IRD1 expression from the additional copy of the Gtf2ird1 allele (i.e., the additional copy of Gtf2ird1 is a functional null).

Development

The Gtf2i duplication allele (Gtf2i^Dup; 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 Gtf2i^3'UTRloxP allele (Gtf2i^tm1Lro) 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 Gtf2i^3'UTRloxP mice were bred at least ten generations with CD1, and then used as described below.

The XS0608 allele (Gtf2ird1^{Gt(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 Gtf2i^Dup allele (Dp(5Gtf2i-Gtf2ird1)1Lro), Dr. Osborne first bred CD1.Gtf2i^3'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.Gtf2i^3'UTRloxP::Sycp1-Cre double mutant was then bred to CD1.XS0608 mice; producing the triple mutant (CD1.Gtf2i^3'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 Gtf2i^3'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 Gtf2i^Dup allele to offspring.

Gtf2i^Dup 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 Gtf2i^3'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.

Allele

Symbol: Dp(5Gtf2i-Gtf2ird1)1Lro
Name: duplication, Chr 5, L Osborne 1
MGI accession ID: MGI:5490970
Generation method: Targeted
Attributes: Not Applicable
Marker symbol: Dp(5Gtf2i-Gtf2ird1)1Lro
Marker name: duplication, Chr 5, L Osborne 1
Chromosome: 5
Strain of origin: 129
Molecular note: Cre-mediated recombination between loxP sites in intron 4 of Gtf2ird1^{Gt(XS0608)Wtsi} and downstream of exon 34 of Gtf2i^tm1Lro produced a duplication of the intervening sequence. Western blot analysis confirmed increased expression of Gtf2i but not Gtf2ird1 in the brain.
General note: Gtf2ird1^{Gt(XS0608)Wtsi} was generated in the E14.1TGa.4 ES cell line, derived from 129P2/OlaHsd, and Gtf2i^tm1Lro was made in the R1 ES cell line, derived from (129X1/SvJ x 129S1/Sv)F1-Kitl⁺.