B6;SJL-Reck^em2Nat/J

Stock number: 033395
Product name: Reck CC4

Research areas: Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Neurobiology Research

Description

Reck CC4 mice carry CRISPR/Cas9-generated P256A and W261A mutations that eliminate Wnt7a signaling by the Reck (reversion-inducing-cysteine-rich protein with kazal motifs) gene, but do not affect protein folding or trafficking to the plasma membrane. This strain is useful in studies of central nervous system (CNS) angiogenesis and the blood brain barrier (BBB).

Detailed description

Reck (reversion-inducing-cysteine-rich protein with kazal motifs) encodes a multi-domain glycosylphosphatidylinisotol (GPI)-anchored plasma membrane protein with matrix metalloproteinase (MMP) inhibitory activity. The gene plays an essential role in embryonic and placental vascular remodeling. New evidence demonstrates that it is also part of a cell surface protein complex that promotes mammalian central nervous system (CNS) angiogenesis through activation of the canonical Wnt pathway. Reck has also been found to contribute to the regulation of blood brain barrier (BBB) development and maintenance.

This CRISPR-edited Reck CC4 line carries a targeted mutation in the coding region of the Reck gene, resulting in P256A and W261A mutations in the Reck CC4 domain. Homozygous embryos die at approximately embryonic day 9 (~E9). P256 and W261 are essential for Wnt7a-specific canonical signaling in cell culture, and the alanine substitution mutations P256A and W261A eliminate signaling but do not affect protein folding or trafficking to the plasma membrane.

A compound heterozygous combination of the Reck CC4 CRISPR allele with a hypomorphic Reck allele missing exon 2 shows severe defects in CNS angiogenesis, consistent with a defect in Wnt7a and Wnt7b signaling.

Expression of the protein encoded by the Reck CC4 CRISPR allele was observed in compound heterozygous embryos at E11. It is indistinguishable from wildtype in size and abundance.

Development

This CRISPR-edited mouse was generated using an in vitro transcribed guide RNA (sgRNA) targeting the Reck gene. A single-stranded oligodeoxynucleotide (ssODN) template was used for homology directed repair (HDR). The sgRNA, ssODN template, and in vitro transcribed Cas9 mRNA were injected into C57BL/6J x SJL F2 embryos. Resultant mice carrying alanine substitution mutations P256A (nucleotide change: CCT to GCA) and W261A (nucleotide change: TGG to GCC) were backcrossed to C57BL/6J for one generation by the donating lab.

Allele

Symbol: Reck^em2Nat
Name: endonuclease-mediated mutation 2, Jeremy Nathans
MGI accession ID: MGI:6283835
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: Reck^P256A,W261A
Marker symbol: Reck
Marker name: reversion-inducing-cysteine-rich protein with kazal motifs
Marker synonyms: St15
Chromosome: 4
Strain of origin: (C57BL/6J x SJL/J)F2
Molecular note: CRISPR/Cas9 genome editing is used to insert alanine substitution mutations P256A (nucleotide change: CCT to GCA) and W261A (nucleotide change: TGG to GCC). The mutations eliminate Wnt7a signaling by the Reck gene, but do not affect protein folding or trafficking to the plasma membrane.