These OVE2497B-SB2 (OVE#2497B-SB2) mice harbor a mutation created by random insertion of the SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223), and the transposon was subsequently mobilized via exposure to sleeping beauty transposase (SB10) to generate the mutation. Using inverse PCR analysis, the transposon integration site was identified in intron 10 of the protein kinase, cGMP-dependent, type I gene (Prkg1) on chromosome 19. The IR/DR is linked to the (-) strand of DNA at position 30,692,871 bp [NCB137/mm9; 30,692,871(-)]. The rtTA is inserted in the sense orientation relative to the disrupted mouse gene.

The donating investigator reports the phenotype of homozygous mice as: post-weaning lethal.

A lentiviral transgenic approach was used to generate these mice. The SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223) was designed with a Sleeping Beauty (SB) transposon (containing a slice-acceptor::IRES::rtTA::polyA gene trap), a mouse tyrosinase minigene (Tyro), and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in the FUGW self-inactivating HIV-based lentiviral vector backbone. The SB transposon and Tyro minigene replaced the ubiquitin-c promoter and EGFP sequences originally found in the FUGW lentiviral vector. The 1200 bp SB transposon used in this transgene has an inverted repeat/direct repeat sequence (IR/DR; the SB transposon recognition site), an adenovirus splice acceptor, a stop sequence (3xSTOP), an internal ribosome entry site (IRES; from human X-chromosome-linked inhibitor of apoptosis (XIAP)), a sequence encoding an optimized form of reverse tetracycline controlled transactivator (rtTA2S; with stop codon), a human growth hormone polyA sequence, and a second IR/DR sequence. The IR/DR sequences are outward-facing (pointed away from the sa-IRES-rtTA-pA). The Tyro minigene is composed of the mouse Tyr enhancer region (623 bp), promoter region (657 bp), and 1566 bp cDNA sequence (including the stop codon); all in sense orientation relative to the FUGW backbone. There is no polyA site between the Tyro minigene and WPRE sequence. The WPRE sequence functions to enhance the mRNA transcript stability. This packaged lentiposon was injected subzonally (under the zona pellucida) into 1-8 cell stage FVB/N mouse embryos. Expression of the tyrosinase minigene results in melanin synthesis, so founder mice (F0) were identified by inspection for pigmentation. All F0 mice exhibited mosaic, light, pigmentation. Most F0 mice had more than one lentiviral integration site. F0 mice were assigned an OVE number and then bred with FVB/N mice. Pigmented offspring (F1) with different coat colors were designated as subline A, B, C, etc., for each family. F1 mice were bred with FVB/N mice. F2 mice with different coat colors were designated as sublines A-1, A-2, etc., or B-1, B-2, etc. Breeding with FVB/N mice was continued until litters with only one or two different coat colors were obtained. Mice with identical coat colors were then inbred to generate homozygotes. Homozygous mice were viable and fertile. Homozygous females were bred to males with testes-specific expression of the SB transposase (PGK2-SB10 transgenic line OVE1780 on an FVB/N genetic background). The resulting double mutant male offspring ("seed males") have the ability to mobilize transposons in their germline; and mobilization of the outward-facing, IR/DR site-flanked transposon results in integration at new sites in the genome. The seed males were bred with FVB/N wildtype females, and the offspring were screened for presence of the original lentiviral insertion site (positive for tyrosinase) and for transposition of the splice-acceptor::IRES::rtTA::polyA transposon. Mice with transpositions (new F0 mice) were bred to FVB/N mice to establish new lines (SB1, SB2, etc.). If the new integration site is still linked to the original lentiviral integration site, the mice are pigmented. If the original lentiviral integration has been bred away from the new integration site, the mice are albino. F1 mice for each new line were inbred and assessed for the presence or absence of viable homozygotes. Hemizygous mice of line OVE2497B-SB2 (OVE#2497B-SB2) exhibit medium grey coat color. The donating investigator reports the PGK2-SB10 transgene was bred out of the line as well. Using inverse PCR analysis, the transposon integration site was identified in intron 10 of the protein kinase, cGMP-dependent, type I gene (Prkg1) on chromosome 19. The IR/DR is linked to the (-) strand of DNA at position 30,692,871 bp [NCB137/mm9; 30,692,871(-)]. The rtTA is inserted in the sense orientation relative to the disrupted mouse gene. Transgenic males were sent to The Jackson Laboratory Repository node of the Mutant Mouse Regional Resource Center (MMRRC-JAX). Upon arrival, these mice were bred to FVB/NJ inbred mice (Stock No. 001800) for at least one generation to establish the live colony.

Wild-type littermates or FVB/NJ

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact csmmrrc@jax.org. Older strains may not have this information.

Paul A. Overbeek, Ph.D., Baylor College of Medicine