B6(Cg)-Notch3^{tm1.1(NOTCH3)Arbo}/J

Stock number: 040785
Product name: hNotch3 cKO [loxP-huNOTCH3-IRES-GFP-pA-loxP-mCherry-pA]
Research areas: Cardiovascular Research, Developmental Biology Research, Internal/Organ Research, Research Tools

Description

hNOTCH3 conditional knock-out (cKO) mice have a loxP-flanked human NOTCH3 cDNA and enhanced green fluorescent protein variant (EGFP) sequence, followed by a red fluorescent protein variant sequence (mCherry), inserted into exon 1 of the mouse Notch3 locus, abolishing endogenous Notch3 expression. Prior to Cre-mediated recombination, these mice will express the humanized NOTCH3 protein and EGFP in Notch3-expressing cells. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have the human NOTCH3 and EGFP sequences deleted in cre-expressing tissues, allowing only expression of the mCherry fluorescent protein. These mice may be useful when studying the development and maintenance of vascular smooth muscle cells as well as vascular disorders, such as CADASIL.

Detailed description

Notch3 protein is a transmembrane receptor that plays a role in regulating the function and survival of mural cells including vascular smooth muscle cells and pericytes. Notch3 mutations are the primary cause of CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), which presents with recurrent strokes, cognitive decline, and white matter lesions. These hNOTCH3 conditional knock-out (cKO) mice express a loxP-flanked human NOTCH3 cDNA and enhanced green fluorescent protein variant (EGFP) sequence, followed by a red fluorescent protein variant sequence (mCherry). The loxP–hNotch3 cDNA–IRES–EGFP–PolyA–loxP–mCherry–PolyA vector was inserted into the ATG start site of the mouse Notch3 gene, abolishing endogenous Notch3 expression.

Homozygous mice are viable and fertile with no overt phenotype. Prior to Cre-mediated recombination, these mice express the humanized NOTCH3 protein and EGFP in Notch3-expressing cells. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have the human NOTCH3 and EGFP sequences deleted in cre-expressing tissues, allowing only expression of the mCherry fluorescent protein. EGFP and mCherry fluorescence are detectable by immuno-staining and direct fluorescence. These mice may be useful when studying the development and maintenance of smooth muscle cells in the vasculature system, as well as when studying the vascular disorder CADASIL.

Development

The hNOTCH3 conditional knock-out (cKO) targeting vector was designed to insert loxP sites flanking a human NOTCH3 cDNA and an enhanced green fluorescent protein variant (EGFP) sequence, followed by a red fluorescent (mCherry) gene all inserted into exon 1 of the mouse Notch 3 (Notch3) locus. The full targeting construct consisted of an initial loxP site, human NOTCH 3 (NOTCH3) cDNA, an internal ribosome entry site (IRES) sequence, an enhanced green fluorescent protein (EGFP), polyadenylation tail (polyA), an FRT-flanked neomycin cassette, a second loxP site an mCherry sequence and a second polyadenylation tail (polyA). The construct was electroporated into C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric mice were bred with a FLP-recombinase mouse line to remove the FRT-flanked neomycin cassette. hNOTCH3 cKO mice, with neomycin cassette removed, were maintained on a C57BL/6 genetic background by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Notch3^{tm1.1(NOTCH3)Arbo}
Name: targeted mutation 1.1, Joseph Arboleda-Velasquez
MGI accession ID: MGI:8220372
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Null/Knockout; Inserted expressed sequence; Humanized sequence
Marker symbol: Notch3
Marker name: notch 3
Marker synonyms: CADASIL; CADASIL1; CARASIL1; CASIL; FPLD1; hpbk; IMF2; LMNS; N3
Chromosome: 17
Strain of origin: C57BL/6
Molecular note: The targeting vector was designed to insert loxP sites flanking a human NOTCH3 cDNA and an enhanced green fluorescent protein (EGFP) sequence, followed by a red fluorescent (mCherry) gene all inserted into exon 1 of the mouse Notch 3 (Notch3) at the ATG start site, abolishing endogenous Notch3 expression. The full targeting construct consisted of an initial loxP site, human NOTCH 3 (NOTCH3) cDNA, an internal ribosome entry site (IRES) sequence, an enhanced green fluorescent protein (EGFP), polyadenylation tail (polyA), an FRT-flanked neomycin cassette, a second loxP site an mCherry sequence and a second polyadenylation tail (polyA). A FLP-recombinase removed the FRT-flanked neomycin cassette.