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 and assessed for the presence or absence of viable homozygotes. Hemizygous mice of line OVE2495A (OVE#2495A) exhibit light mottle coat color. Using inverse PCR analysis, the lentiviral integration site was identified in intron 5 of the splicing factor 3a, subunit 1 gene (Sf3a1) on chromosome 11. The 5'-LTR is linked to the (+) strand of DNA at position 4,071,241 bp [NCB137/mm9; 5'-4,071,241(+)]. The rtTA is inserted in the sense orientation relative to the disrupted mouse gene. Transgenic males were used to freeze sperm, and that cryopreserved sperm were sent to The Jackson Laboratory Repository node of the Mutant Mouse Regional Resource Center (MMRRC-JAX). Upon arrival, aliquots of this frozen sperm were used to fertilize oocytes from FVB/NJ inbred females to establish the live colony.
