NOD.Cg-Gt(ROSA)26Sor^em5Mvw/MvwJ

Stock number: 036181
Product name: NOD.RosaBxb-GT/GA
Research areas: Research Tools

Description

NOD.RosaBxb-GT/GA mice are a resource for the rapid creation of vector-free targeted alleles in the Gt(ROSA)26Sor locus on the NOD/ShiLtJ background. The dual Bxb1 attP-site "safe harbor landing pad" in the first intron of Gt(ROSA)26Sor enables efficient and precise integration of a donor DNA sequence by recombinase mediated cassette exchange (RMCE), and the vector backbone sequence is simultaneously excluded.

Of note: The Bxb-GT/GA dual landing pad platform is available on the C57BL/6J congenic background as Stock No. 036152, on the C57BL/6NJ congenic background as Stock No. 039420, on a B6 albino background as Stock No. 036153, on the NSG as Stock No. 036151, and on the NOD/ShiLtJ background as Stock No. 036181.

Detailed description

This strain provides a tool for precise, site-specific insertion of single-copy sequence into the Gt(ROSA)26Sor intron 1-2 in a manner that is in the desired orientation and free from vector sequence. In practice, donor DNA with a desired sequence flanked by the requisite attB sites is delivered along with the Bxb1 integrase (mRNA or protein) into the pronucleus of fertilized oocytes containing at least one duel landing pad allele. The result is a unidirectional, single-copy integration of the sequence into the Gt(ROSA)26Sor locus at high efficiency. 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 Bxb-GT/GA dual landing pad allele utilizes heterologous attachment sites (attP-GA and attP-GT) which increases the integration efficiency as much as 3-fold over that observed using homologous sites. Additionally, this system permits the site-specific insertion of significantly larger (~30kb) constructs than some other systems (Xu et al., 2013). Moreover, as few pseudo-sites exist in the mouse genome which are recognized by Bxb1 integrase, this system maintains exceptionally high fidelity site-specific insertion in the targeted locus with low probability of off-target effects.

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

Of note: The Bxb-GT/GA dual landing pad platform is available on the C57BL/6J congenic background as Stock No. 036152, on the C57BL/6NJ congenic background as Stock No. 039420, on a B6 albino background as Stock No. 036153, on the NSG as Stock No. 036151, and on the NOD/ShiLtJ background as Stock No. 036181.

Development

The RosaBxb-GT/GA 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. Guide RNAs were selected to target intron 1 of the Gt(ROSA)26Sor locus on Chromosome 6. Oligonucleotide donor DNAs were created encoding a duel landing pad flanked by the heterologous attP attachment sites - specifically a 48 bp Bxb1 attP-GT sequence, a 240 bp spacer of native Gt(ROSA)26Sor sequence and a 48 bp Bxb1 attP-GA sequence. These sequences and Cas9 nuclease were introduced into NSG zygotes (Stock No. 005557), which were transferred into pseudopregnant females. Progeny were screened by DNA sequencing to identify correctly targeted pups. Founder mice were then bred to NSG mice for germline transmission and then intercrossed to generate homozygotes (available as Stock No. 036151). Some mice were then backcrossed to NOD/ShiLtJ mice (Stock No. 001976) to remove the Prkdc^scid and Il2rg^tm1Wjl alleles, then intercrossed to generate homozygotes.

The NOD.RosaBxb-GT/GA strain (Stock No. 036181) was generated by backcrossing a female NOD.Cg-Gt(ROSA)26Sor^em5Mvw Prkdc^scid Il2rg^tm1Wjl/MvwJ (NSG.RosaBxb-GT/GA; Stock No. 036151) to NOD/ShiLtJ (Stock No. 001976) then backcrossing a male from the resulting litter to NOD/ShiLtJ females and selectively intercrossing to exclude the Prksc^scid and Il2rg^tm1Wjl alleles from this subline.

Allele

Symbol: Gt(ROSA)26Sor^em5Mvw
Name: endonuclease-mediated mutation 5, Michael Wiles
MGI accession ID: MGI:6160836
Generation method: Endonuclease-mediated
Attributes: RMCE-ready
Synonyms: NSG.Bxb1v2; NSG.RosaBxb-GT/GA
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: NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ
Molecular note: CRISPR/cas9 genome editing used guide RNAs to target intron 1 of the Gt(ROSA)26Sor locus. Oligonucleotide donor DNAs were created encoding a duel landing pad flanked by the heterologous attP attachment sites - specifically a 48 bp Bxb1 attP-GT sequence, a 240 bp spacer of native Gt(ROSA)26Sor sequence and a 48 bp Bxb1 attP-GA sequence.