B6.Cg-Gt(ROSA)26Sor^{tm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj} Trp53^tm1Brn/TyjJ

Stock number: 037954
Product name: Rosa26^PE2;Trp53^flox
Strain synonyms: Rosa26^LSL-PE2;Trp53^flox
Research areas: Neurobiology Research, Research Tools

Description

Rosa26^PE2 Trp53^flox knock-in mice allow cre-inducible expression of a prime editor (PE2) allele consisting of a human optimized cas9 fused to a MMLV-RT (Moloney murine leukemia virus reverse transcriptase, GAG-POL) and an mNeonGreen reporter in the context of a cre-mediated Trp53 null allele. The Rosa26^PE2 Trp53^flox model facilitates in vivo prime editing for precise editing of transition and transversion single nucleotide variants (SNVs) and indels in murine tissues and in vitro prime editing in organoids and cell lines derived from various tissues. This strain may be used to accelerate preclinical functional studies of cancer-associated alleles and complex genetic combinations.

Of note, a strain carrying only the Rosa26^PE2 allele is distributed as B6.Cg-Gt(ROSA)26Sor^{tm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj}/J (Stock No. 037953).

Detailed description

Rosa26^PE2 Trp53^flox knock-in mice conditionally express a prime editor (PE2) allele consisting of a human optimized Cas9 with the H840A mutation fused to a MMLV-RT (Moloney murine leukemia virus reverse transcriptase, GAG-POL) and an mNeonGreen reporter from the Gt(ROSA)26Sor locus and carry a conditional floxed Trp53 allele. The PE2 enzyme complexes with prime editing guide RNAs that encode a mutation of interest within a reverse transcriptase template. The construct is preceded by a lox-stop-lox (LSL) allowing for cre-inducible expression. Mice homozygous for both mutations are viable and fertile. The Rosa26^PE2 Trp53^flox model facilitates in vivo prime editing for precise editing of transition and transversion single nucleotide variants (SNVs) and indels in murine tissues without the need for exogenous delivery of a prime editor enzyme. This enables tumor formation driven by specific cancer-associated mutations, in combination with a cre-mediated Trp53 null allele. The model also facilitates in vitro prime editing in organoids and cell lines derived from various tissues. The model can be used to create the broad spectrum of cancer-associated mutations, combinations of mutations and genetic lesions. The donating investigator reports that mNeonGreen is visible by direct green fluorescence; it is robust and bright in in vivo tumors and in vitro organoids, and dim in fibroblasts but still visible under the microscope.

Of note, a strain carrying only the Rosa26^PE2 allele is distributed as B6.Cg-Gt(ROSA)26Sor^{tm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj}/J (Stock No. 037953).

Development

The ROSA26 LSL PE2 targeting construct contains from 5' to 3': a CAGG promoter, FRT site, loxP-stop-loxP, a human optimized Cas9 with the H840A mutation fused to a MMLV-RT (Moloney murine leukemia virus reverse transcriptase, GAG-POL), P2A peptide, an mNeonGreen reporter, WPRE, polyA signal, and a PGK-FRT3-NeoR-polyA. The cre-inducible cas9*/ GAG-POL fusion construct is referred to as the prime editor enzyme (PE2). The PE2 complexes with prime editing guide RNAs that can encode a mutation of interest within a reverse transcriptase template. The mNeonGreen reporter is one of the brightest green/yellow fluorescent proteins and is derived from Branchiostoma lanceolatum. The FRT/FRT3 sites allows Flp-mediated replacement of the central construct (Rosa26^PE2) for the use of future prime editor enzymes. Of note, the donating investigator has not tested the Flp-mediated recombination. The entire construct was inserted into the Gt(ROSA)26Sor locus on chromosome 6 via electroporation of B6.129P2-Trp53^tm1Bern derived KP4 embryonic stem (ES) cells.

The Trp53^flox targeting vector contains flanking loxP sites to introns 1 and 10 of the Trp53 locus on chromosome 11. An IB10/E14IB10 129P2/OlaHsd-derived embryonic stem cell line was used to create the mutation. This line was obtained from Dr. Anton Berns (University of Amsterdam) and backcrossed to C57BL/6 for eight generations by the donating laboratory of Dr. Tyler Jacks to create the mouse strain, B6.129P2-Trp53^tm1Brn/Tyj.

Correctly targeted ES cells for the LSL-PE2 allele were selected, and chimeric males were bred to C57BL/6J females by the donating laboratory (see SNP note). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. One of the 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.