STOCK Vim^{tm2(flpo)Ggen}/J

Stock number: 039724
Product name: Vim^{FLEX(SA-H2B-eGFP-T2a-FlpO-WPRE-pA)}
Strain synonyms: Vim^Flpo, Vim-GFP-FlpO
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA)} mice have inward facing lox2272 sites and loxP sites flanking exons 2-4 of the Vim gene and an inverted cassette containing a Flpo-recombinase (flpo), T2A sequence, enhanced green fluorescent protein (EGFP), and a human HIST1H2BB gene [histone 1 H2bb]. These Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA)} mice express endogenous wildtype VIM prior to Cre-mediated recombination. Following Cre-mediated recombination, exons 2-4 are removed and the H2B1-EGFP-FLPo recombinase knock-in cassette is inverted into the correct orientation for expression from the Vim promoter. This strain may be useful for studying the role of VIM in cell migration, cell shape and plasticity, and Epithelial-to-Mesenchymal Transition (EMT).

The donating laboratory has also made a Cre-inducible Vim^{FLEX(SA-eGFP-T2A-HSV-TK-WPRE-pA)} mouse line available as Stock No. 039691.

Detailed description

Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA)} mice have a lox2272 site upstream of exon 2 of the Vimentin (Vim) gene and an inverted cassette flanked with inward facing loxP sites downstream of exon 4. The cassette contains a polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a Flpo-recombinase (flpo), T2A sequence, enhanced green fluorescent protein (EGFP), a human HIST1H2BB gene [histone 1 H2bb], a splice acceptor site and a second lox2272 site in opposite orientation to the first, followed by an FRT-flanked neomycin cassette and a second inward facing loxP site. Vimentin is a structural protein expressed by mesenchymal cells and plays a role in cell migration, cell shape and plasticity, and Epithelial-to-Mesenchymal Transition (EMT). These Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA)} mice express endogenous wildtype VIM prior to Cre-mediated recombination. Following Cre-mediated recombination, exons 2-4 are removed and the H2B-EGFP-FLPo recombinase knock-in cassette is inverted into the correct orientation for expression from the Vim promoter.

Homozygous Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA)} mice are viable and fertile with no overt phenotype. Following Cre-mediated recombination, EGFP is detectable by immunofluorescence and fluorescence-activated cell sorting (FACS).

The donating laboratory has also made a Cre-inducible Vim^{FLEX(SA-eGFP-T2A-HSV-TK-WPRE-pA)} mouse line available as Stock No. 039691.

Development

The Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA} targeting vector was designed with (from 5'to 3') an inward facing lox2272 site upstream of exon 2 of the vimentin(Vim) gene, followed by exons 3 and 4, and an inverted cassette flanked with inward facing loxP sites. The inverted floxed cassette contains a polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a Flpo-recombinase (flpo), T2A sequence, enhanced green fluorescent protein (EGFP), a human HIST1H2BB gene [histone 1 H2bb], a splice acceptor site and a second lox2272 site in opposite orientation to the first, followed by an FRT-flanked neomycin cassette and a second inward facing loxP site all downstream of exon 4. This construct was electroporated into C57BL/6N-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred to Flp-deleter mice to remove the Neo cassette, and then to C57BL/6N animals for 10 generations. The donating laboratory further bred these Vim^{FLEX(SA-H2B-eGFP-T2A-FlpO-WPRE-pA} with a R26-lsl-TdTomato (Stock No. 007905) and an H11-lsl-spCas9 (Stock No. 026816). Prior to arrival at The Jackson Laboratory, both the R26-lsl-TdTomato and H11-lsl-spCas9 alleles were removed. Upon arrival sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

In 2024, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. All of the 43 markers throughout the genome were C57BL/6-derived. Three of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Vim^{tm2(flpo)Ggen}
Name: targeted mutation 2, Giannicola Genovese
MGI accession ID: MGI:7718776
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Recombinase-expressing; Reporter
Synonyms: Vim^{FLEX(SA-H2B-eGFP-T2a-FlpO-WPRE-pA)}; Vim^FlpO
Marker symbol: Vim
Marker name: vimentin
Chromosome: 2
Strain of origin: C57BL/6N
Molecular note: The targeting vector was designed with (from 5'to 3') an inward facing lox2272 site upstream of exon 2 of the vimentin(Vim) gene, followed by exons 3 and 4, and an inverted cassette flanked with inward facing loxP sites. The inverted floxed cassette contains a polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a Flp-recombinase (FlpO), T2A sequence, enhanced green fluorescent protein (EGFP), a human HIST1H2BB gene [histone 1 H2bb], a splice acceptor site and a second lox2272 site in opposite orientation to the first, followed by an FRT-flanked neomycin cassette and a second inward facing loxP site all downstream of exon 4.