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 (containing a cHS4 core insulator element), 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), a 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 (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 lentiposon 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#2323C were bred with FVB/N mice, and pigmented offspring (F1) were bred to FVB/N mice. The resulting offspring (F2) with identical coat colors (dark grey) were subsequently inbred. Using inverse PCR analysis, the transgene integration site was identified in intron 3 of the PHD finger protein 5A gene (Phf5a) on chromosome 15 (specifically at the 5'-81,697,556(+) 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.
