A lentiviral transgenic approach was used to generate these mice. The Tyro-WPRE-FUGW lentiviral transgene (LV2177) was designed with a mouse tyrosinase minigene (Tyro) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in the FUGW self-inactivating HIV-based lentiviral vector backbone. The Tyro minigene replaced the ubiquitin-c promoter and EGFP 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 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 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#2295G-2b3 were bred with FVB/N mice, and pigmented offspring (F1) were bred to FVB/N mice. The resulting offspring (F2) with identical coat colors (medium grey) were subsequently inbred. Using inverse PCR analysis, the transgene integration site was identified in intron 3 of the ATP-binding cassette, sub-family E, member 1 gene (Abce1) on chromosome 8 (specifically at the 3'-82,228,217(-) bp position in sense 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.
