C57BL/6J-Sema5a^em1Rojg/J

Stock number: 039227
Product name: Sema5a^GAG
Strain synonyms: Sema5a (R747E_R749E)
Research areas: Developmental Biology Research, Neurobiology Research

Description

Sema5a^GAG knock-in (KI) mice carry two CRISPR/Cas9-mediated point mutations in the Sema5a gene, resulting in disruption of Semaphorin-5A binding to heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs). These mice may be useful for studying the role of Sema5A:(GAG) interactions in neuronal development and neurodevelopmental disorders, such as autism spectrum disorder.

Detailed description

Semaphorins are a large family of secreted, transmembrane, or GPI-anchored proteins that contain an extracellular sema-domain and variable juxta membrane domains. Semaphorins have been implicated in regulation of cytoskeletal dynamics, adhesion, and migration in both neuronal and non-neuronal cell types and are necessary for tissue development. Semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects toward developing axons through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for binding GAGs.

Sema5a^GAG mice harbor a CRISPR/Cas9-generated mutant allele with two point-mutations in the Sema5a gene. These mutations result in an arginine to glutamic acid substitution at residues 747 and 749 (R747E_R749E), leading to disruption of Sema5A binding to HS-GAGS and CS-GAGS.

Homozygous Sema5a^GAG mice are viable and fertile with no overt phenotype. While Sema5A protein levels in brain tissue are comparable between wildtype and Sema5a^GAG mice, the mutant mice demonstrate defects in hippocampal dentate gyrus progenitor cell migration. The mutation also influences PlxnA2 mediated progenitor cell distribution in the dentate. These mice may be useful for studying the role of Sema5A:glycosaminoglycan (GAG) interactions in neuronal development and neurodevelopmental disorders, such as autism spectrum disorder.

Development

The Sema5a^GAG knock-in allele was generated using CRISPR/Cas9 endonuclease-genome editing. A guide RNA [5'-gatccaaatttgctggaagtagg-3'] was selected to introduce two point-mutations (AGAcagAGG>GAAcagGAG) in the sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A (Sema5a) locus on chromosome 15. Sema5a transcript Sema5a-201 (ENSMUST00000067458.7) was used as reference. Cas9 nuclease mRNA, sgRNAs and a DNA donor template sequence were introduced into the cytoplasm of zygotes derived from C57BL/6J (Stock No. 000664) mice with well recognized pronuclei. Correctly targeted pups were identified by PCR and Sanger sequencing. The resulting founder mice were crossed to C57BL/6J mice for 2 generations to reduce off-target CRISPR changes. 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 oocytes (Stock No. 000664).

Donor DNA template (ssODN) used to generate this model is as follows:
5'- TGAGCAGCGTTTCCGCTATACCTGTAAAGCTCGCCTGCCAGATCCAAATTTGCTcGAgGTcGGAgaACAGgaGATAGAAATGCGGTACTGTTCCAGCGATGGAACCAGTGGCTGCTCCACAGA -3'.

Allele

Symbol: Sema5a^em1Rojg
Name: endonuclease-mediated mutation 1, Roman J Giger
MGI accession ID: MGI:7618232
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: Sema5a (R747E_R749E); Sema5a^GAG
Marker symbol: Sema5a
Marker name: sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A
Marker synonyms: 9130201M22Rik; M-Sema D; SEMAF; semF
Chromosome: 15
Strain of origin: C57BL/6J
Molecular note: Using CRISPR/Cas9mediated gene editing, nucleotide substitution leading to amino acids R747E and R749E point mutations were introduced to glycosaminoglycan (GAG) binding sites.