B6.Cg-Gt(ROSA)26Sor^em7Mvw Tyr^c-2J/MvwJ

Stock number: 036153
Product name: B6A.RosaBxb-GT/GA
Strain synonyms: B6A.RosaBxb-GT/GA
Research areas: Dermatology Research, Mouse/Human Gene Homologs, Research Tools, Sensorineural Research

Description

B6A.RosaBxb-GT/GA mice are a resource for the rapid creation of vector-free targeted alleles in the Gt(ROSA)26Sor locus on the albino C57BL/6J 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

B6A.RosaBxb-GT/GA mice have the dual Bxb1 attP-site "safe harbor landing pad" in the first intron of Gt(ROSA)26Sor. This is designed to allow site-specific and precise insertion of a donor DNA sequence that is free from vector sequence, does not require the use of homology arms and integrates as a single copy at high efficiency.

This Bxb-GT/GA dual landing pad allele utilizes heterologous attachment sites (attP-GT and attP-GA) which increases the integration efficiency as much as 3-fold over that observed using a single attachment site. [Low et al., 2022]

In practice, a donor DNA vector is prepared containing the corresponding Bxb1 attB sites flanking up to or greater than ~40kb of desired DNA sequence (e.g., Addgene plasmids 175390 or 175391). The donor plasmid is then delivered along with the Bxb1 integrase (mRNA or protein) into the pronucleus of fertilized oocytes from this RosaBxb-GT/GA strain for recombinase mediated cassette exchange (RMCE). This is designed to result in highly efficient unidirectional, single-copy integration of the donor sequence into the Gt(ROSA)26Sor locus in up to ~50% of offspring. The inserted sequence will be flanked by the recombined attachment sites, now called Bxb1 attR(GT) and Bxb1 attL(GA), but will be otherwise scar-less (free from residual vector sequence from the plasmid backbone). [Low et al., 2022]

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 B6A.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. First, a single Guide RNA was selected to target intron 1 of the Gt(ROSA)26Sor locus on Chromosome 6 and a 48bp dsDNA donor (with no homology arms) was used to integrate the Bxb1 attP-GT site into the resulting dsDNA break. These reagents were introduced into C57BL/6J zygotes (Stock No. 000664) and, after an initial backcross to C57BL/6J, the resulting progeny were intercrossed to generate homozygous embryos which were targeted again to insert a second heterologous attP-GA Bxb1 site. The attP-GA sequence was delivered as a ssDNA oligonucleotide donor (with asymmetric homology arms) along with Cas9 and sgRNA to position this site 240bp 3' of the first site. The end result is a 48 bp Bxb1 attP-GT sequence and a 48 bp Bxb1 attP-GA sequence separated by a 240 bp spacer of native Gt(ROSA)26Sor sequence.

Microinjected embryos were transferred into pseudopregnant females and progeny were screened by PCR to identify correctly targeted pups. Sequencing revealed a small indel (-3bp/+6bp) in the Gt(ROSA)26Sor locus immediately 5' of the Bxb1 attP-GT sequence which is predicted to have no impact on the function of the allele. A single founder mouse was backcrossed to C57BL/6J mice for germline transmission and the verified offspring were interbred to establish a homozygous colony. After re-targeting to add the second site, a single founder mouse was again backcrossed to C57BL/6J mice for germline transmission and the resulting offspring were interbred to generate Stock No. 036152. Next, some mice were bred to B6-albino (Stock No. 000058 B6(Cg)-Tyr^c-2J/J), and the pups were bred together to be homozygous for both alleles. The resulting B6A.RosaBxb-GT/GA mice were sent to The Jackson Laboratory Repository as Stock No. 036153.

Allele

Symbol: Gt(ROSA)26Sor^em7Mvw
Name: endonuclease-mediated mutation 7, Michael Wiles
MGI accession ID: MGI:6757188
Generation method: Endonuclease-mediated
Attributes: RMCE-ready
Synonyms: B6.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: C57BL/6J
Molecular note: Using CRISPR-Cas9 and oligo-mediated homology directed repair, two heterologous attachment sites, Bxb1 attP-GT and Bxb1 attP-GA, were inserted into intron 1-2, providing cognate attachment and recombination sites for Bxb1 attB-GT and Bxb1 attB-GA, for unidirectional, single-copy donor transgenic integration mediated by Bxb1 Integrase.

Allele

Symbol: Tyr^c-2J
Name: albino 2 Jackson
MGI accession ID: MGI:1855985
Generation method: Spontaneous
Attributes: Null/Knockout
Marker symbol: Tyr
Marker name: tyrosinase
Marker synonyms: ATN; CMM8; Oca1; OCA1A; OCAIA; SHEP3; skc35
Chromosome: 7
Strain of origin: B6.Cg-Tyrp1^b Hps1^ep
Site of expression: Expressed in melanosomes
Molecular note: This mutation has a G-to-T base change at coding nucleotide 230 resulting in an amino acid change from arginine to leucine at residue 77 (p.R77L) which lies in the highly conserved DDRE sequence. Nucleotide 230 is at the alternative 5' splice donor site for exon 1 and this allele does not produce the 1a or 1b subset of tyrosinase transcripts but does produce a significant increase in 1c and 1d transcripts. Western blots of homozygous mutant skin extracts demostrate the nearly complete absence of the broad 76-84 kDa band of glycosylated wild-type tyrosinase. No tyrosinase activity was found in hairbulb extracts from homozygous mice.