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

Stock number: 037953
Product name: Rosa26^PE2 , LSL PE2 , Rosa26^LSL-PE2
Research areas: Neurobiology Research, Research Tools

Description

Rosa26^PE2 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. The Rosa26^PE2 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 the Rosa26^PE2 allele and a floxed Trp53 allele is distributed as B6.Cg-Gt(ROSA)26Sor^{tm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj} Trp53^tm1Brn/TyjJ (Stock No. 037954).

Detailed description

Rosa26^PE2 knock-in mice conditionally express a prime editor enzyme (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. 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. Homozygous mice are viable and fertile. The Rosa26^PE2 model facilitates in vivo prime editing to create transition and transversion single nucleotide variants (SNVs) and indels in murine tissues without the need for exogenous delivery of a prime editor enzyme. The model also facilitates in vitro prime editing in organoids and cell lines derived from various tissues. The model can be used to create a broad spectrum of disease-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 the Rosa26^PE2 allele and a floxed ^Trp53 allele is distributed as B6.Cg-Gt(ROSA)26Sor^{tm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj} Trp53^tm1Brn/TyjJ (Stock No. 037954).

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 primer 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^tm1Brn derived KP4 embryonic stem (ES) cells. Correctly targeted ES cells were selected, and chimeric males were bred to C57BL/6J females. 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). Once established the Trp53^tm1Brn allele was bred out of the colony.