FVB/N-In(6Exoc4)2Ove/Mmjax

Stock number: 017402
Research areas: Developmental Biology Research

Description

These OVE2346B-SB3a (OVE#2346B-SB3a) mice harbor a mutation created by random insertion of the SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229), 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: flattened "pancake" appearance at embryonic day (E)9.

Detailed description

These OVE2346B-SB3a (OVE#2346B-SB3a) mice harbor a mutation created by random insertion of the SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229), and the transposon was subsequently mobilized via exposure to sleeping beauty transposase (SB10) to generate the mutation. Using inverse PCR analysis, a chromosomal inversion was identified on chromosome 6 between the original lentiviral integration site (intron 10 of the exocyst complex component 4 gene (Exoc4; linked to the (+) strand of DNA at position 33,571,516 bp [NCB137/mm9; 3'-33,571,516(+)]) and the transposition site (intron 10 of Exoc4; the right IR/DR is linked to the (+) strand of DNA at position 33,531,556 bp [NCB137/mm9; R3-33,531,556(+)]). The donating investigator reports the phenotype of homozygous mice as: flattened "pancake" appearance at embryonic day (E)9.

Development

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 (inward-facing SB arms flanking a tandem pair of cHS4 core insulator elements), followed by 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), two copies of the 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 (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 lentiposon 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 to generate homozygotes. Homozygous mice were viable and fertile. Homozygous females were bred to males 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 from the inward-facing, IR/DR configuration leads to genomic rearrangements including deletions, inversions and translocations, in conjunction with loss of the cHS4 core insulator element initially located between the IR/DR elements. The seed males were bred with FVB/N wildtype females, and the offspring were screened for deletion of the core insulator element. Mice with transpositions (new F0 mice) were bred to FVB/N mice to establish new lines (SB1, SB2, etc.). F1 mice for each new line were inbred and assessed for the presence or absence of viable homozygotes. Hemizygous mice of line OVE2346B-SB3a (OVE#2346B-SB3a) exhibit medium grey mottle coat color. The donating investigator reports the PGK2-SB10 transgene was bred out of the line as well. Using inverse PCR analysis, a chromosomal inversion was identified on chromosome 6 between the original lentiviral integration site (intron 10 of the exocyst complex component 4 gene (Exoc4; linked to the (+) strand of DNA at position 33,571,516 bp [NCB137/mm9; 3'-33,571,516(+)]) and the transposition site (intron 10 of Exoc4; the right IR/DR is linked to the (+) strand of DNA at position 33,531,556 bp [NCB137/mm9; R3-33,531,556(+)]). Transgenic males were sent to MMRRC-JAX. Upon arrival, these mice were bred to FVB/NJ for at least one generation to establish the colony.

Allele

Symbol: In(6Exoc4)2Ove
Name: inversion, Chr 6, Paul A Overbeek 2
MGI accession ID: MGI:5287033
Generation method: Transposon induced
Marker symbol: In(6Exoc4)2Ove
Marker name: inversion, Chr 6, Paul A Overbeek 2
Chromosome: 6
Strain of origin: FVB/N
Molecular note: The SB-cHS4core-SB-Tyro-WPRE-FUGW lentiposon transgene (LV2229) was designed with a Sleeping Beauty (SB) transposon (inward-facing SB arms flanking a tandem pair of cHS4 core insulator elements), followed by 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), two copies of the 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 (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 transposon was mobilized by SB10. In line OVE2346B-SB3a, an inversion occurs at the lentiposon integration site in intron 10 of Exoc4 [NCBI37/mm9; 3'-33,571,516(+) and 33,531,556(+)].