C57BL/6J-Ctnna1^em2Cgot/Mmjax

Stock number: 041099
Product name: Ctnna1^4A
Strain synonyms: Ctnna1 PKI-4A
Research areas: Cancer Research, Cell Biology Research, Developmental Biology Research

Description

Ctnna1^4A mice harbor 4 amino acid (S641A, S652A, S655A, T658A) substitutions in exon 14 of the Ctnna1 gene. The introduced mutations prevent the phosphorylation of α-E-catenin. These mice may be useful when studying the regulatory role of α-E-catenin phosphorylation in actomyosin contractility as well as cell junction structure and function.

Detailed description

Ctnna1 (catenin alpha 1) gene encodes a mechanosensitive cell junction protein, α-E-catenin, which is one of the essential actin-binding proteins of the cadherin/catenin adhesion complex involved in cell-cell adhesion. There are multiple isoforms of α-catenins encoded by different genes; Ctnna1 is ubiquitously expressed and essential for development and homeostasis of most tissues. α-E-catenin is abnormally expressed in leukemia and other cancers such as breast, gastric and genitourinary. These Ctnna1^4A mice harbor 4 amino acid (S641A, S652A, S655A, T658A) substitutions in exon 14 of the Ctnna1 gene. The introduced mutations prevent the phosphorylation of α-E-catenin.

Heterozygous mice are viable and fertile, though they are slightly smaller in body size/weight compared to wildtype controls. Homozygous mice are sub-viable, with lower than expected Mendelian ratios from heterozygous x heterozygous matings. Surviving homozygous mice demonstrate runted appearance, hydrocephaly, curly/bent tails and a lifespan of ~8-10 weeks. Ctnna1^4A mice may be useful when studying the regulatory role of α-E-catenin phosphorylation in actomyosin contractility as well as cell junction structure and function.

Development

The Ctnna1^4A allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. Guide RNAs (G19F 5'-TTAATACGACTCACTATAGGGGCTGCAGACCCCCGAGGAGTGTTTTAGAGCTAGAAATAGCAAGT-3' and G21F 5'-TTAATACGACTCACTATAGGGTCAGCTGGTCATCTTCTGTCGTTTTAGAGCTAGAAATAGCAAGT-3'), were used to target exon 14 of the catenin alpha 1 (Ctnna1) gene on chromosome 18. Bolded nucleotides correspond to sequences in Ctnna1. Ctnna1 transcript Ctnna1-201 (ENSMUST00000042345.8) was used as reference for exon number and guide sequences. The gRNAs, donor DNA containing the 4 codon mutations (S641A, S652A, S655A, T658A) in exon 14 of the Ctnna1 gene, and Cas9 nuclease were introduced into the cytoplasm of zygotes derived from C57BL/6J mice with well recognized pronuclei. No founders harboring all 4 substitutions were generated from this first round injections. However, one male harbored 3 of the 4 mutations (CK1 sites, S652A, S655A, T658) and was bred to C57BL/6J females to establish a mouse line. To create the 4th mutation (CK2 site, S641A), the G19F gRNA (G19F 5'-TTAATACGACTCACTATAGGGGCTGCAGACCCCCGAGGAGTGTTTTAGAGCTAGAAATAGCAAGT-3'), along with donor DNA containing the S641A mutation and Cas9 nuclease were electroporated into the cytoplasm of zygotes derived from homozygous mice harboring the S652A, S655A, T658 mutations. Two founder lines, 1034 and 1035, were generated harboring all 4 substitutions. Founder line 1035 was further expanded and backcrossed to C57BL/6J mice for at least 10 generations by the donating laboratory. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Ctnna1^em2Cgot
Name: endonuclease-mediated mutation 1, Cara Gottardi
MGI accession ID: MGI:8270340
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: Ctnna1^4A
Marker symbol: Ctnna1
Marker name: catenin (cadherin associated protein), alpha 1
Marker synonyms: 2010010M04Rik; alpha E catenin; alpha(E)-catenin; CAP102; catenin (cadherin associated protein), alpha 1; Catna1; MDBS2; MDPT2
Chromosome: 18
Strain of origin: C57BL/6J
Molecular note: Using CRISPR/Cas9 endonuclease-mediated genome editing, a guide RNA (G19F 5'-TTAATACGACTCACTATAGGGGCTGCAGACCCCCGAGGAGTGTTTTAGAGCTAGAAATAGCAAGT-3') was used to target exon 14 of the catenin alpha 1 (Ctnna1) gene to introduce the codon mutations (S641A) in exon 14 in the Ctnna1^em1Cgot allele harboring 3 of the 4 mutations (CK1 sites, S652A, S655A, T658).