A lentiviral transgenic approach was used to generate these mice. The Tyro-sd-IRES-loxP-FUGW lentiviral transgene (LV2187) was designed with a mouse tyrosinase minigene (Tyro) followed by a splice-donor::IRES::loxP sequence in the FUGW self-inactivating HIV-based lentiviral vector backbone. The Tyro-sd-IRES-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 (620 bp), promoter region (500 bp), and 1.7 kbp cDNA sequence (including the stop codon); all in antisense orientation relative to the FUGW backbone. The Tyro minigene is followed by an artificial splice donor sequence, an internal ribosome entry site (IRES), and a loxP site in this construct. This packaged lentiviral construct was injected subzonally (under the zona pellucida) into 1-8 cell stage FVB/N mouse embryos. Because of the random and multiple lentiviral integration sites of a transgene containing tyrosinase, founder mice (F0) may be identified by mosaic pigmentation. Founder mice from line OVE#2416B were bred with FVB/N mice, and pigmented offspring (F1) were bred to FVB/N mice. The resulting offspring (F2) with identical coat colors (light brown) were subsequently inbred. Using inverse PCR analysis, the transgene integration site was identified in intron 5 of the WEE 1 homolog 1 gene (Wee1) on chromosome 7 (specifically at the 5'-117,273,626(+) bp position in antisense orientation). Transgenic males were sent to The Jackson Laboratory Repository (MMRRC-JAX). Upon arrival, these mice were bred to FVB/NJ inbred mice for at least one generation to establish the live colony.
