STOCK Nup62^{Tg(Tyr)2397E-1a2Ove}/Mmjax

Stock number: 017382
Research areas: Developmental Biology Research

Description

These OVE#2397E-1a2 mice harbor a mutation created by random insertion of the Tyro-sd-IRES-loxP-FUGW lentiviral transgene (LV2187). Using inverse PCR analysis, the transgene integration site was identified in intron 1/exon 2 of the nucleoporin 62 gene (Nup62) on chromosome 7 (specifically at the 5'-52,083,776(+) bp position in antisense orientation). The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)7 lethal.

Detailed description

These OVE#2397E-1a2 mice harbor a mutation created by random insertion of the Tyro-sd-IRES-loxP-FUGW lentiviral transgene (LV2187). Using inverse PCR analysis, the transgene integration site was identified in intron 1/exon 2 of the nucleoporin 62 gene (Nup62) on chromosome 7 (specifically at the 5'-52,083,776(+) bp position in antisense orientation). The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)7 lethal.

Development

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#2397E-1a2 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 1/exon 2 of the nucleoporin 62 gene (Nup62) on chromosome 7 (specifically at the 5'-52,083,776(+) 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 (see SNP results below).

A 27 SNP (single nucleotide polymorphism) panel analysis, with markers covering all 19 chromosomes and the X chromosome, was performed on the rederived living colony at The Jackson Laboratory Repository. This revealed 3 of 27 markers, one each on chromosomes 11, 13 and X, that were segregating for allele-type markers other than FVB/N. These data suggest the mice are ~89% FVB/N genetic background with a contribution not specifically described here.

Allele

Symbol: Nup62^{Tg(Tyr)2397E-1a2Ove}
Name: transgene lentiviral insertion 2397E-1a2, Paul A Overbeek
MGI accession ID: MGI:5286652
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Nup62
Marker name: nucleoporin 62
Chromosome: 7
Strain of origin: FVB/N
Expressed genes: Tyr,tyrosinase,mouse, laboratory
Molecular note: The Tyro-IRES-sd-loxP-FUGW lentiviral transgene (LV2187) was designed with a mouse tyrosinase minigene (Tyro) followed by an IRES::3xATG::splice-donor::loxP sequence in the FUGW self-inactivating HIV-based lentiviral vector backbone. The Tyro-IRES-sd-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 (623 bp), promoter region (657 bp), and 1566 bp cDNA sequence (including the stop codon); all in antisense orientation relative to the FUGW backbone. The Tyro minigene is followed by an internal ribosome entry site (IRES) ending with ATGs in all three reading frames (3xATG), an artificial splice donor sequence, and a loxP site in this construct. In line 2397E-1a2, the lentiviral transgene inserted into intron 1/exon 2 (NCBI37/mm9; 5'-52,083,776(+)) in antisense orientation relative to the disrupted gene.