These Vcam1ΚB mice express a mutant VCAM1 with an inactivated NF-ΚB binding site. They are suitable for applications related to the study of the role of VCAM1 NF-ΚB binding in atherosclerosis, acute inflammation and systemic endothelial activation.
David S Milstone, Brigham and Women's Hospital
The Vcam1 gene encodes a cytokine-inducible endothelial adhesion molecule that regulates leukocyte rolling and arrest on endothelial cells. The molecule plays a key role in the inflammation associated with atherosclerosis and rheumatoid arthritis.
These mice carry the Vcam1NFkB5'mut allele containing mutant sequence (6 nucleotides in the transcriptional promoter and 73 to 64 nucleotides 5’ of the transcription start site) in the NF-kB binding site domain region, as well as incidental 11-13 nucleotides 5' from the translational start. A 3 nucleotide sequence substitution was introduced into the 5' UTR to distinguish the mutant and wildtype mRNA and hnRNA. Mice that are homozygous for the targeted mutation are viable and fertile, but produce smaller litter sizes. Reduced expression of the mutant gene (mRNA or protein) is detected by Northern or Western blot analysis of heart tissue from LPS challenged homozygotes. In mutant homozygous mice immunized with ovalbumin, leukocyte recruitment in immune mediated peritonitis is reduced.
Sequential tag and exchange gene targeting was used to introduce a 6 nucleotide mutation in the 5’NF-ΚB binding site of the transcriptional promoter, and a 3 nucleotide mutation in the 5’UTR of Vcam1 in exon 1. The first, “Tag”, targeting vector was used to replace exon 1 and part of 2 with PGK-NEO/TK cassettes.
The second, “exchange”, vector was utilized to introduce the 6 nucleotide mutation in the 5’ NF-ΚB binding site, and a 3 nucleotide sequence substitution was introduced into the 5' UTR, which distinguishes the mutant and wildtype mRNA and hnRNA.
The first construct was electroporated into 129S4/SvJae derived J1 embryonic stem (ES) cells.
A correctly targeted clone of ES cells was then transfected with the second targeting vector containing both the 6 nucleotide mutation in the 5’NF-ΚB binding site and the 3 nucleotide mutation in the 5’UTR region. ES cells carrying the desired Vcam1ΚB allele were injected into C57BL/6J and BALB/cJ blastocysts. The resulting chimeric male animals were crossed to C57BL/6J mice, and then backcrossed to C57BL/6J for a total of at least 10 generations.
Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.
|Allele Name||targeted mutation 2, David Milstone|
|Allele Type||Targeted (Not Applicable)|
|Gene Symbol and Name||Vcam1, vascular cell adhesion molecule 1|
|Strain of Origin||129S4/SvJae|
|Molecular Note||Sequential tag and exchange gene targeting was used to introduce a 6 nucleotide mutation in the 5NF-B binding site of the transcriptional promoter, and a 3 nucleotide mutation in the 5UTR of Vcam1 in exon 1. The first, "tag", targeting vector replaces exon 1 (encoding the signal peptide) and part of exon 2 (encoding immunoglobulin-like domain 1) with a pgk-neomycin/TK cassettes. The Tag construct is transfected into embryonic stems cells and a single correctly targeted clone is transfected with the second construct.The second "Exchange" targeting construct replaces the neo cassette and introduces a 6 nucleotide substitution 5' of the transcription start site resulting in the disruption of the 5'NFKB1 (NF-kappaB) binding site domain and a 3 nucleotide substitution downstream of the translation start site in the 5' UTR of exon 1. The 3 nt substitution is used to distinguish the mutant and wild-type mRNA and hnRNA. Western blot ananlysis demonstrates the presence of mutant protein in heart endothelial cells.|
When maintaining a live colony, these mice can be bred as homozygotes. Homozygotes are viable and fertile, but have smaller litter sizes.
When using the Vcam1 NFkB5'mut mouse strain in a publication, please cite the originating article(s) and include JAX stock #027679 in your Materials and Methods section.