FVB;B6-Cript^{Tn(sb-rtTA)2427.SB1Ove}/Mmjax

Stock number: 017393
Research areas: Developmental Biology Research

Description

These OVE#2427-SB1 mice harbor a mutation created by random insertion of the SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223), and the transposon was subsequently mobilized via exposure to sleeping beauty transposase (SB10) to generate the mutation. The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)8 lethal.

Detailed description

These OVE#2427-SB1 mice harbor a mutation created by random insertion of the SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223), and the transposon was subsequently mobilized via exposure to sleeping beauty transposase (SB10) to generate the mutation. Using inverse PCR analysis, the transposon integration site was identified in intron 3 of the cysteine-rich PDZ-binding protein gene (Cript) on chromosome 17 (specifically at the R3-87,432,586(+) bp position with the rtTA in sense orientation relative to the endogenous gene). The donating investigator reports the phenotype of homozygous mice as: embryonic day (E)8 lethal.

Development

A lentiviral transgenic approach was used to generate these mice. The SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223) was designed with a Sleeping Beauty (SB) transposon (containing a slice-acceptor::IRES::rtTA::polyA gene trap), 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), an adenovirus splice acceptor, an internal ribosome entry site (IRES; from human X-chromosome-linked inhibitor of apoptosis (XIAP)), a reverse tetracycline controlled transactivator sequence (rtTA; with stop codon), a human growth hormone polyA sequence, and a second IR/DR sequence. The IR/DR sequences are outward-facing (pointed away from the sa-IRES-rtTA-pA). 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 C57BL/6 mouse embryos. Founder mice (F0) with random, multiple lentiviral integration sites were identified by PCR. Founder mice from line OVE#2427-SB1 were identified and bred with FVB/N mice. Pigmented offspring were bred to mice with testes-specific expression of the SB transposase (PGK2-SB10 transgenic line OVE1780 on an FVB/N genetic background). The resulting double mutant 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 integration at new sites in the genome. The seed males were bred with FVB/N wildtype females. The resulting transgenic mice harbor the original integration(s) of the Tyro-FUGW transgene, but new integration(s) of the splice-acceptor::IRES::rtTA::polyA transposon. If the new integration site is still linked to the original lentiviral integration site, the mice are pigmented. If the original lentiviral integration has been bred away from the new integration site, the mice are albino. Offspring with identical coat colors (dark grey) were subsequently inbred. The donating investigator reports the PGK2-SB10 transgene was bred out of the line as well. Using inverse PCR analysis, the transposon integration site was identified in intron 3 of the cysteine-rich PDZ-binding protein gene (Cript) on chromosome 17 (specifically at the R3-87,432,586(+) bp position with the rtTA in sense orientation relative to the endogenous gene). 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.

Allele

Symbol: Cript^{Tn(sb-rtTA)2427.SB1Ove}
Name: transposon insertion 2427.SB1, Paul A Overbeek
MGI accession ID: MGI:5287502
Generation method: Transposon induced
Marker symbol: Cript
Marker name: cysteine-rich PDZ-binding protein
Chromosome: 17
Strain of origin: C57BL/6
Molecular note: A lentiviral transgenic approach was used to generate these mice. The SB-sa-IRES-rtTA-pA-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2223) was designed with a Sleeping Beauty (SB) transposon (containing a slice-acceptor::IRES::rtTA::polyA gene trap), 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 1200 bp SB transposon used in this transgene has an inverted repeat/direct repeat sequence (IR/DR; the SB transposon recognition site), an adenovirus splice acceptor, a stop sequence (3xSTOP), an internal ribosome entry site (IRES; from human X-chromosome-linked inhibitor of apoptosis (XIAP)), a sequence encoding an optimized form of reverse tetracycline controlled transactivator (rtTA2S; with stop codon), a human growth hormone polyA sequence, and a second IR/DR sequence. The IR/DR sequences are outward-facing (pointed away from the sa-IRES-rtTA-pA). 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. The splice-acceptor::IRES::rtTA::polyA transposon is mobilized by testes-specific expression of the SB transposase. In line OVE2427-SB1, the transposon integrated into intron 3 [NCBI37/mm9; R3-87,432,586(+)] in the sense orientation.