FVB/N-Uba3^{Tg(Tyr)2295C-2Ove}/Mmjax

Stock number: 017373
Research areas: Developmental Biology Research

Description

These OVE#2295C-2 mice harbor a mutation created by random insertion of the Tyro-WPRE-FUGW lentiviral transgene (LV2177). Using inverse PCR analysis, the transgene integration site was identified in exon 1/intron 2 of the ubiquitin-like modifier activating enzyme 3 gene (Uba3) on chromosome 6 (specifically at the 3'-97,153,331(+) bp position in antisense orientation). The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)6 lethal.

Detailed description

These OVE#2295C-2 mice harbor a mutation created by random insertion of the Tyro-WPRE-FUGW lentiviral transgene (LV2177). Using inverse PCR analysis, the transgene integration site was identified in exon 1/intron 2 of the ubiquitin-like modifier activating enzyme 3 gene (Uba3) on chromosome 6 (specifically at the 3'-97,153,331(+) bp position in antisense orientation). The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)6 lethal.

Development

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 (623 bp), promoter region (657 bp), and 1566 bp 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 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 OVE2295C-2 (OVE#2295C-2) exhibit light grey mottle coat color. Using inverse PCR analysis, the lentiviral integration site was identified in exon 1/intron 2 of the ubiquitin-like modifier activating enzyme 3 gene (Uba3) on chromosome 6. The 3'-LTR is linked to the (+) strand of DNA at position 97,153,331 bp [NCB137/mm9; 3'-97,153,331(+)]. The lentivirus is inserted in the antisense orientation relative to the disrupted mouse gene. Transgenic males were used to freeze sperm, and that cryopreserved sperm were sent to The Jackson Laboratory Repository node of the Mutant Mouse Regional Resource Center (MMRRC-JAX). Upon arrival, aliquots of this frozen sperm were used to fertilize oocytes from FVB/NJ inbred females to establish the live colony.

Allele

Symbol: Uba3^{Tg(Tyr)2295C-2Ove}
Name: transgene lentiviral insertion 2295C-2, Paul A Overbeek
MGI accession ID: MGI:5287495
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Uba3
Marker name: ubiquitin-like modifier activating enzyme 3
Chromosome: 6
Strain of origin: FVB/N
Expressed genes: Tyr,tyrosinase,mouse, laboratory
Molecular note: 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 (623 bp), promoter region (657 bp), and 1566 bp 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. In line OVE2295C-2, the lentivirus integrates into exon 1/intron 1 [NCBI37/mm9; 3'-97,153,331(+)] in the antisense orientation.