This "safe harbor landing pad" allele on the 129S1/SvImJ 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). [<a href="https://pubmed.ncbi.nlm.nih.gov/35361849/">Low et al., 2022</a>] 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.

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RosaBxb-GT mice are a resource for the rapid creation of vector-free targeted alleles in the Gt(ROSA)26Sor locus on the 129S1/SvImJ 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.

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