B6.129-Vil1^tm1Gum/J

Stock number: 028946
Product name: Vil-lacZ knock-in/knock-out
Research areas: Cancer Research, Internal/Organ Research, Research Tools

Description

The Vil-LacZ knock-in/knockout allele (also called Villin^β-gal) was designed to both abolish Vil1 gene function and express a fusion transcript encoding the 18 N-terminal amino acids of villin fused in-frame to the β-galactosidase cDNA. Expression of lacZ is directed to villus and crypt epithelial cells, along the entire small intestine and colon, in a cephalocaudal gradient similar to the expression pattern of endogenous Vil1. These mice may be useful in studying intestinal organogenesis, gastric stem cell biology and cancer.

Detailed description

In adult mice, the villin 1 gene (Vil1) is highly expressed specifically in the intestinal epithelial cells, whereas the gastric epithelium is largely devoid of villin. The Vil-LacZ knock-in/knockout allele (also called Villin^β-gal) has lacZ replacing exons 1-3 of the Vil1 locus. This abolishes endogenous gene function and results in the villin 1 promoter/enhancer regions directing expression of a fusion transcript encoding the 18 N-terminal amino acids of villin fused in-frame to the β-galactosidase cDNA. No villin protein expression from this null allele is observed in intestinal epithelial tissue. Homozygous villin-deficient mice are viable and fertile with no gross abnormalities. Subtle ultrastructural alterations in the microvillar actin core of the intestine are reported in homozygotes. Villin/lacZ expressing cells are easily detectable by X-gal staining, both in whole embryos and in sections. Specifically, lacZ expression is primarily in villus and crypt epithelial cells, along the entire small intestine and colon, in a cephalocaudal gradient similar to the expression pattern of endogenous Vil1. Additionally, a rare population of gastric progenitor cells (GPCs) in the antrum are lacZ positive in an otherwise lacZ negative adult gastric epithelium.

Development

The Vil-LacZ knock-in/knockout allele (also called Villin^β-gal) was designed by Dr. Deborah L. Gumucio (University of Michigan). A targeting vector was designed to replace exons 1-3 of the villin 1 locus (Vil1) with a cDNA encoding β-galactosidase (lacZ) and a reverse-oriented PGK-neomycin resistance cassette. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺)-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6J females for germline transmission. The donating investigator reports that the mutant colony was maintained by breeding heterozygous males with C57BL/6NCrl females for more than ten generations (see SNP results below). In 2016, heterozygous males with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6 background during backcrossing.

In 2016, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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 a cohort of the first generation rederived living colony at The Jackson Laboratory Repository. 26 of 27 markers throughout the genome suggested a C57BL/6 genetic background - the only exception was the marker in the panel closest to the Vil1 gene (segregating C57BL/6;129S allele-type). In addition, 4 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (chromosomes 8 [~15.2 Mbp], 13 [~41.0 Mbp], 15 [~57.6 Mbp] and 19 [~49.9 Mbp]). These data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background.

Allele

Symbol: Vil1^tm1Gum
Name: targeted mutation 1, Deborah L Gumucio
MGI accession ID: MGI:2676462
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Vil1
Marker name: villin 1
Chromosome: 1
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: lacZ,beta-galactosidase,E. coli
Site of expression: Gastric progenitor cells.
Molecular note: A targeting construct containing lacZ and neo replaced 4.6 kb of genomic sequence encompassing a portion of the first coding exon (exon 1) and the following 2 exons (exons 2 and 3). An upstream non-translated exon was left intact. While low levels of aberrant transcipt were detected in mutant mice, neither normal nor truncated protein was detected by Western blot analysis.