B6.Cg-Gt(ROSA)26Sor^{tm2.1(CAG-APOBEC1/YTHDF2*)Kmey}/J

Stock number: 040011
Product name: DARTmut
Strain synonyms: APOBEC1-YTHmut
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

APOBEC1-YTH^mut knock-in (KI) mice harbor a floxed stop cassette followed by a fusion protein sequence consisting of a mutant human m⁶A-binding YTH domain fused to the rat cytidine deaminase APOBEC1 and an HA-tag, knocked-into the Gt(ROSA)26Sor locus. The YTH^mut fusion lacks amino acids 385-409 comprising the m⁶A-binding region.

These mice may be used as a control for the APOBEC1-YTH KI mouse line (Stock No. 040005) when evaluating RNA methylation (m⁶A) in cre-expressing cells and tissues.

Detailed description

APOBEC1-YTH^mut knock-in (KI) mice express a Cre-inducible HA-tagged, fusion protein consisting of a mutant human m⁶A-binding YTH domain, which lacks amino acids 385-409, fused to a rat cytidine deaminase (APOBEC1), from the Gt(ROSA)26Sor locus. This APOBEC1-YTHmut protein exhibits diminished binding to m6A. The N⁶-methyladenosine (m⁶A) modification has been identified as a major post-transcriptional regulatory mark in different types of RNAs and plays a role in regulating RNA splicing, translation, stability, translocation and structure. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will express the human APOBEC1-YTH^mut protein in cre-expressing cells and tissues. Homozygous APOBEC1-YTH^mut KI mice are viable and fertile with no overt phenotype. These mice may be used as a control for the APOBEC1-YTH KI mouse line (Stock No. 040005) when evaluating RNA methylation (m⁶A) in cre-expressing cells and tissues by facilitating the identification of false-positive C-to-U editing events.

Development

The APOBEC1-YTH^mut knock-in (KI) allele was generated by cloning the APOBEC1-YTH-HA sequence from pCMV-APOBEC1-YTH (Addgene, 131636) into the GT(ROSA)26Sor locus (Ai9 Addgene, 22799). The final targeting construct consists of a CAG (CMV enhancer/chicken beta-actin) promoter, a loxP-flanked STOP cassette, a rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1) sequence fused to a human mutant YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) sequence, followed by an HA tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. The construct was electroporated into (129S6/SvEvTac X C57BL/6NCr)F1-derived G4 embryonic stem (ES) cells and correctly targeted ES cells were injected into recipient blastocysts. The chimeric mice were crossed to a codon-optimized phiC31 mouse line (Stock No. 007670) to remove the neo cassette from the germline. The resulting APOBEC1-YTH KI mice, with neo cassette removed, were backcrossed to C57BL/6J for at least 5 generations by the donating laboratory and then maintained by crossing to an UBC-Cre^ER mouse line (Stock No. 007001) (see SNP note below). APOBEC1-YTH^mut KI mice provided to The Jackson Laboratory were wildtype for the UBC-Cre^ER allele. Upon arrival, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

In 2025, 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 43 markers throughout the genome was segregating with an unknown source on Chromosome 13. Also, 4 of the 5 markers that determine C57BL/6J from C57BL/6N were 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.

Allele

Symbol: Gt(ROSA)26Sor^{tm2.1(CAG-APOBEC1/YTHDF2*)Kmey}
Name: targeted mutation 2.1, Kate D Meyer
MGI accession ID: MGI:7783574
Generation method: Targeted
Attributes: Inserted expressed sequence
Synonyms: APOBEC1-YTH^mut
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: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting construct consists of a CAG (CMV enhancer/chicken beta-actin) promoter, a loxP-flanked STOP cassette, a rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1) sequence fused to a human mutant YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) sequence, followed by an HA tag, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette was inserted into the GT(ROSA)26Sor locus. FLP-recombinase removed the neo cassette.