B6N.Cg-Stra8^em1Dcp/J

Stock number: 034072
Product name: Stra8^FLAG
Research areas: Research Tools

Description

The long, meiosis-critical isoform of the mouse Stra8 (stimulated by retinoic acid gene 8) gene is FLAG-tagged in these Stra8^FLAG mice, enabling biochemical analyses.

Detailed description

Stra8 (stimulated by retinoic acid gene 8) is a germ cell-specific gene required for meiotic initiation. It additionally upregulates a transcriptional network of thousands of genes whose expression is not limited to meiosis. The mouse Stra8 gene encodes two isoforms, the longer of which is critical for meiotic initiation. The shorter isoform, lacking the first 111 amino acids, is not able to initiate meiosis.

These Stra8^FLAG mice express an N-terminal (exon 2) FLAG tag knock-in that labels only the longer, meiosis-critical isoform of the mouse Stra8 gene. Expression of Stra8 is unimpeded. Anti-FLAG antibody effectively detects the functional FLAG-tagged protein, enabling biochemical analyses. Chromatin immunoprecipitation sequencing (ChIP-seq), using anti-FLAG antibody to immunoprecipitate testicular chromatin, has demonstrated that STRA8 protein binds directly to genomic regulatory regions.

Heterozygous and homozygous Stra8^FLAG males and females are viable and fertile; their gonads display normal histology.

Development

The Stra8^Δ121 allele was developed using CRISPR/Cas9 methodologies. One-cell C57BL/6NTac-129S6/SvEvTac hybrid embryos were injected with a mixture of gRNA, Cas9, and a 200 bp single-stranded oligo encoding a FLAG peptide and an 8 amino acid linker (sequence GSGSGSGS), flanked on both sides by approximately 75 bp of exon 2 sequence.

Two mutant alleles were generated in this process: this Stra8^FLAG allele, expressing a FLAG-tagged long-isoform protein (Stock No. 034072) and the Stra8^Δ121 long-isoform knock-out allele (see Stock No. 034073), carrying a 121 bp deletion in exon 2 instead of a FLAG insertion. The Stra8^Δ121 allele continues to express the short isoform of Stra8.

Stra8^FLAG mutant mice were backcrossed to C57BL/6NTac for at least 10 generations by the donating laboratory. Upon arrival at The Jackson Laboratory, C57BL/6NJ (Stock No. 005304) was used for strain maintenance.

Allele

Symbol: Stra8^em1Dcp
Name: endonuclease-mediated mutation 1, David C Page
MGI accession ID: MGI:6303961
Generation method: Endonuclease-mediated
Attributes: Reporter; Epitope tag
Marker symbol: Stra8
Marker name: stimulated by retinoic acid gene 8
Chromosome: 6
Strain of origin: C57BL/6 x 129
Molecular note: Using an sgRNA and CRISPR/Cas9 technology, a FLAG peptide and an 8-aa linker sequence was inserted directly after the ATG in exon 2 of Stra8 gene.