A Sleeping Beauty (SB) transposon transgenic approach was used to generate these mice. The BART6 transposon vector used here was designed by Dr. Colin E. Bishop (Baylor College of Medicine).
This BART6 transposon vector is the same as the pT2/BART3 transposon vector, and has (from 5' to 3') an inverted repeat/direct repeat sequence (IR/DR; the SB transposon recognition site [280 bp]), an AD2 splice acceptor and polyA sequence, the tyrosinase minigene TyBS, a loxP site, a polyA sequence, a Hox9c-based splice acceptor, and right IR/DR (280 bp). The IR/DR sequences are outward-facing. The 4.1 kb tyrosinase minigene (TyBS) contains the tyrosinase upstream regulatory sequences (2.1 kbp from the BALB/c tyrosinase promoter and the first 65 bp of exon 1), followed by the C57BL/6-derived Tyrs-J cDNA sequence (1.9 kbp including cysteine at amino acid 103, glycine at amino acid 346, and the stop codon with polyA signal).
This transgene was microinjected into one-cell stage FVB/N embryos. Expression of the tyrosinase minigene results in melanin synthesis, and founder mice (F0) were identified by inspection for pigmentation. F0 mice were identified and then bred with FVB/N mice.
Pigmented offspring (F1) with different coat colors were designated as individual sublines. F1 mice were bred with FVB/N mice. This continued until mice with identical coat colors were obtained, and then bred together to generate homozygotes. Homozygous mice were bred to mice with widespread expression of the SB transposase (CAGGS-SB10 transgenic mice on an FVB/N genetic background). The resulting double transgenic male offspring ("seed males") have the ability to mobilize transposons in their germline; and mobilization of the outward-facing, IR/DR site-flanked transposon results in transposon integration at new sites in the genome (as well as genomic rearrangements including deletions, inversions and translocations).
Seed males were bred with FVB/N females, and offspring were screened for new coat colors (i.e.; new integration sites of the transposon). Mice with new transpositions were bred to FVB/N mice to establish new lines (TP1, TP2, etc.). Mice for each new line were inbred and assessed for phenotype. Mice from line BART6-TP1H had congenital absence of both eyes (anophthalmia). Heterozygous and homozygous mice from this line exhibit light grey/medium grey coat color. These BART6-TP1H mice were then sent to Dr. Paul Overbeek (Baylor College of Medicine) for further characterization.
There, the original transgene integration site was not able to be precisely mapped, but was found near the bone morphogenetic protein 4 locus (Bmp4) on mouse chromosome 14. The transposed integration site is reported to be at 46,829,514 [NCB137/mm9] on chromosome 14: this is ~150 kb away from Bmp4 (47,003,195-47,010,344 [NCB137/mm9]). The transposed integration site is tightly linked with the original integration site.
Dr. Overbeek reports the CAGGS-SB10 transgene was bred out of the line. Transgenic males were then sent to The Jackson Laboratory Repository Facebase collection. Upon arrival, mice were bred to FVB/NJ inbred mice (Stock No. 001800) for at least one generation to establish the colony.
