C57BL/6-Pgbd5^tm2.1Aken/J

Stock number: 039713
Product name: Pgbd5^{3xFlag-HA-P2A-eGFP}
Research areas: Cancer Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

Pgbd5^{3xFlag-HA-P2A-eGFP} knock-in mice express a 3xFLAG-HA tagged endogenous PGBD5 protein, as well as a P2A-self-cleaved EGFP marker under direction of the Pgbd5 promoter. These mice may be useful for tracking Pgbd5 expression.

Detailed description

PiggyBac transposable element derived 5 (PGBD5) is one of the most highly conserved vertebrate transposases which mediates cut-and-paste DNA transposition of piggyBac transposons. PGBD5 is expressed during embryogenesis and neurogenesis, but also found in increased levels in many solid tumors such as medulloblastomas, neuroblastomas, Ewing sarcomas, desmoplastic small round cell tumors, and rhabdomyosarcomas. This Pgbd5^{3xFlag-HA-P2A-eGFP} knock-in (KI) model expresses a FLAG-HA tagged Pgbd5 with a P2A-self-cleaved EGFP marker inserted upstream of stop codon in exon 7 of the Pgbd5 gene.

Homozygous Pgbd5^{3xFlag-HA-P2A-eGFP} KI mice are viable and fertile with no overt phenotype. The donating laboratory confirmed that EGFP expression is detectable in Pgbd5-expressing neurons but not astrocytes or microglia of the dentate gyrus. The donating laboratory also detected certain cell populations of adrenal glands and testes that were EGFP positive. The EGFP signal is detectable by direct fluorescence. These Pgbd5^{3xFlag-HA-P2A-eGFP} KI report mice may be useful for tracking Pgbd5-expressing cells.

Development

The Pgbd5^{3xFlag-HA-P2A-eGFP} knock-in (KI) targeting vector was designed to insert sequence for three FLAG tags (3xFlag), a hemagglutinin (HA) tag, a P2A self-cleaving peptide, followed by an enhanced green fluorescent protein (EGFP) and an Frt-flanked neomycin cassette upstream of the stop codon in exon 7 of the piggyBac transposable element derived 5 (Pgbd5) locus. The construct was electroporated into C57BL/6NTac-derived iTL BF1 embryonic stem (ES) cells (C57BL/6 FLP), which express a FLP recombinase vector to remove the selection cassette. Correctly targeted ES cells were injected into BALB/c blastocysts. The resulting chimeric mice were bred with C57BL/6J mice to remove the Flp-recombinase allele. Pgbd5^{3xFlag-HA-P2A-eGFP} KI mice, with neomycin cassette removed, were maintained on a C57BL/6J genetic background by the donating laboratory (see SNP note below). 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 (Stock No. 000664) oocytes.

In 2024, 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. Three 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: Pgbd5^tm2.1Aken
Name: targeted mutation 2.1, Alex Kentsis
MGI accession ID: MGI:7705048
Generation method: Targeted
Attributes: Reporter; Epitope tag
Synonyms: Pgbd5^{3xFlag-HA-P2A-eGFP}
Marker symbol: Pgbd5
Marker name: piggyBac transposable element derived 5
Marker synonyms: 2900019M05Rik; NEDSHS
Chromosome: 8
Strain of origin: C57BL/6NTac
Molecular note: The targeting vector was designed to insert sequence for three FLAG tags (3xFlag), a hemagglutinin (HA) tag, a P2A self-cleaving peptide, followed by an enhanced green fluorescent protein (eGFP) and an FRT-flanked neomycin cassette upstream of the stop codon in exon 7 of the piggyBac transposable element derived 5 (Pgbd5) locus. Flp-recombinase removed the neomycin cassette.