A/J-Gt(ROSA)26Sor^em18Mvw/Mmjax

Stock number: 037407
Product name: AJ.RosaBxb-GT
Research areas: Research Tools

Description

This strain has been discontinued due to difficulties in cryopreserving strains on A/J genetic background. Please search the JAX Mice database for other similarly-designed RosaBxb-GT strains. As an alternative, researchers may consider obtaining such mice and then backcrossing to A/J inbred mice (Stock No. 000646).

RosaBxb-GT mice are a resource for the rapid creation of vector-free targeted alleles in the Gt(ROSA)26Sor locus on the A/J background. The single Bxb1 integrase attP-site "safe harbor landing pad" in the first intron of Gt(ROSA)26Sor enables efficient and precise integration of a donor DNA sequence directly in zygotes by recombinase-mediated knock-in (RMKI) using vector-free minicircle donor DNA carrying the transgene of interest and the cognate attB-GT attachment site.

Detailed description

This "safe harbor landing pad" allele on the A/J background provides a tool for precise, site-specific insertion of single-copy sequence into the Gt(ROSA)26Sor intron 1 in a manner that is in the desired orientation and free from vector sequence. In practice, a plasmid containing a single cognate 38 bp attB-GT site and the donor sequence is first converted into a vector-less DNA minicircle donor. The DNA minicircle is delivered along with the Bxb1 integrase (mRNA or protein) into the pronucleus of fertilized oocytes containing the 48 bp attP-GT landing pad attachment site. The result is a unidirectional, single-copy integration of the sequence into the Gt(ROSA)26Sor locus. The inserted sequence will be flanked by the recombined attachments sites, now called Bxb1 attL and Bxb1 attR, but will be: (1) free from residual vector sequence from the plasmid backbone, (2) present as a single-copy (no concatemers), and (3) inserted precisely into a known and verifiable locus (Gt(ROSA)26Sor). [Low et al., 2022] The donating investigator indicates that 5% of integrations result in aberrant integration events. In addition, RMKI strategy is successful with inserts up to approximately 15 kb.

Both heterozygous and homozygous mice are viable and fertile with normal breeding, and no reported gross phenotypic or behavioral abnormalities.

Development

The RosaBxb-GT allele was generated in the laboratory of Dr. Michael V. Wiles (The Jackson Laboratory) using CRISPR/Cas9 endonuclease-mediated genome editing/oligo-mediated homology directed repair. An 18-mer TRU-guide RNA was selected to target near the XbaI site of intron 1 in the Gt(ROSA)26Sor locus on Chromosome 6. A 200 nt ssDNA donor (with 76 nt homology arms flanking the 48 bp Bxb1 attP-GT attachment site), the TRU-guide and Cas9 protein were introduced into A/J zygotes by electroporation. Progeny were screened by PCR amplification and DNA sequencing to identify correctly targeted pups. Founder mice were then back-crossed at least once to A/J mice for germline transmission and allele verification before intercrossing to generate homozygotes.

Allele

Symbol: Gt(ROSA)26Sor^em18Mvw
Name: endonuclease-mediated mutation 18, Michael Wiles
MGI accession ID: MGI:6877085
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed); RMCE-ready
Synonyms: A/J.RosaBxb-GT
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: A/J
Molecular note: CRISPR/cas9 mediated HDR using a single stranded oligo, which included homology arms, is used to insert a Bxb1 attP-GT site (48 bp) into intron 1 of the ROSA26 locus.