A lentiviral transgenic approach was used to generate these mice. The SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229) was designed with a Sleeping Beauty (SB) transposon (inward-facing SB arms flanking a tandem pair of cHS4 core insulator elements), followed by 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 SB transposon used in this transgene has an inverted repeat/direct repeat sequence (IR/DR; the SB transposon recognition site), two copies of the 0.25 kb chicken beta-globin core insulator element (cHS4 core), and a second IR/DR sequence. The IR/DR sequences are inward-facing (pointed towards the cHS4core). The cHS4 core insulator element functions to terminate transcription of the upstream locus into which it integrates (and also enhance expression from the downstream Tyro minigene). 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 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 from the inward-facing, IR/DR configuration leads to genomic rearrangements including deletions, inversions and translocations, in conjunction with loss of the cHS4 core insulator element initially located between the IR/DR elements. The seed males were bred with FVB/N wildtype females, and the offspring were screened for deletion of the core insulator element. Mice with transpositions (new F0 mice) were bred to FVB/N mice to establish new lines (SB1, SB2, etc.). F1 mice for each new line were inbred and assessed for the presence or absence of viable homozygotes. Hemizygous mice of line OVE2353A-SB1 (OVE#2353A-SB1) 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, a chromosomal inversion was identified on chromosome 18 between the original lentiviral integration site (agenic region linked to the (-) strand of DNA at position 86,038,197 bp [NCB137/mm9; 3'-86,038,197(-)]) and the transposition site (intron 9 of the Smad4 gene (Smad4); the left IR/DR is linked to the (-) strand of DNA at position 73,805,670 bp [NCB137/mm9; L3-73,805,670(-)]). Transgenic males were used to freeze sperm, and that cryopreserved sperm were sent to MMRRC-JAX. Upon arrival, aliquots of frozen sperm used to fertilize FVB/NJ embryos.
