A lentiviral transgenic approach was used to generate these mice. The Tyro-IRES-sd-loxP-FUGW lentiviral transgene (LV2187) was designed with a mouse tyrosinase minigene (Tyro) followed by an IRES::3xATG::splice-donor::loxP sequence in the FUGW self-inactivating HIV-based lentiviral vector backbone. The Tyro-IRES-sd-loxP replaced the ubiquitin-c promoter, EGFP, and WPRE sequences originally found in the FUGW lentiviral vector. 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 antisense orientation relative to the FUGW backbone. The Tyro minigene is followed by an internal ribosome entry site (IRES) ending with ATGs in all three reading frames (3xATG), an artificial splice donor sequence, and a loxP site in this construct. This packaged lentivirus 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 and assessed for the presence or absence of viable homozygotes. Hemizygous mice of line OVE2372E-3a3 exhibit very light tan/grey coat color and red eyes. Using inverse PCR analysis, the lentiviral integration site was identified in last intron (12) of the kelch-like 20 gene (Klhl20) on chromosome 1. The 3'-LTR is linked to the (-) strand of DNA at position 163,021,329 bp [NCB137/mm9; 3'-163,021,329(-)]. The lentivirus is inserted in the sense orientation relative to the disrupted mouse gene. Transgenic males were later found to exhibit coat color differences. Further investigation revealed a second integration site in intron 2 of the activating transcription factor 7 gene (Atf7) on chromosome 15. After additional breeding, mice harboring only the Klhl20 integration site 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 for at least one generation to establish the live colony.
