B6.129S4(Cg)-Gt(ROSA)26Sor^{tm1(CAG-EGFP*)Bpeng}/J

Stock number: 031413
Product name: RaDR-GFP , B6::RaDR-GFP
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

RaDR-GFP (Rosa26 Direct Repeat-GFP) mice are useful for studying the homologous recombination (HR) double strand break (DSB) DNA repair pathway. They enable in vivo studies of how genetic and environmental factors modulate susceptibility to HR events in disease-relevant tissues (e.g., cancer).
In addition to these C57BL/6J-congenic RaDR-GFP mice (B6::RaDR-GFP ; Stock No. 031413), the donating investigator has also made 129-congenic RaDR-GFP mice available (129::RaDR-GFP ; Stock No. 031299).

Detailed description

DNA damage can lead to toxicity, mutations and ultimately disease - and double strand breaks (DSBs) are acutely damaging. Two major DSB repair pathways used by cells are non-homologous end-joining (NHEJ) which directly rejoins DNA ends, and homologous recombination (HR) which requires a homologous duplex for DSB repair. The correct NHEJ/HR balance is essential for preventing genomic instability.

The Rosa26 Direct Repeat-GFP (RaDR-GFP) allele has a fluorescent direct repeat HR substrate that, upon homologous recombination between the two truncated EGFP expression cassettes, can yield a fluorescent signal. These RaDR-GFP mice enable in vivo studies of how genetic and environmental factors modulate susceptibility to HR events in disease-relevant tissues (e.g., cancer).

Specifically, the GFP direct repeat HR substrate consists of two CAG-ΔEGFP-polyA expression cassettes arranged in tandem (Δ5egfp :: Δ3egfp) - each of which is missing essential sequences of their respective coding sequences: Δ5egfp lacks 15 bp at its 5' end and Δ3egfp lacks 81 bp at its 3' end. Upon homologous recombination between the two truncated EGFP expression cassettes that recombines to encode a functional EGFP sequence, green fluorescence is then observed in those cells. The donating investigator reports that EGFP is visible by direct fluorescence and is much brighter than autofluorescent signals. Under normal conditions, most cells harboring the RaDR-GFP substrate are non-fluorescent, while rare HR events give rise to fluorescent cells (see Sukup-Jackson et al. 2014 PLoS Genet 10:e1004299 - Figure 1B). The donating investigator reports no EGFP fluorescence is observed before HR (although the truncated EGFP proteins are expressed). They also recommend against staining with antibodies, as most antibodies will identify truncated EGFP as well as the full length EGFP.

Both heterozygous and homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.

The donating investigator, Dr. Bevin P. Engelward, has made RaDR-GFP mice are available on a C57BL/6J-congenic background (Stock No. 031413) and a 129-congenic background (Stock No. 031299).

Development

The Rosa26 Direct Repeat-GFP (RaDR-GFP) allele was created in the laboratory of Dr. Bevin P. Engelward (Massachusetts Institute of Technology) to have a PGK-neo-pA cassette followed by a GFP direct repeat HR substrate, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.

The PGK-neo-pA cassette has a single loxP site, the PGK promoter, a neomycin resistance gene (Neo^R) and the PGK polyadenylation signal. The green fluorescent protein (GFP) direct repeat homologous recombination (HR) substrate consists of two CAG-ΔEGFP-polyA expression cassettes arranged in tandem (Δ5egfp :: Δ3egfp). The Δ5egfp cassette has the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an enhanced green fluorescent protein missing 15 bp at its 5' end (Δ5egfp) and a rabbit beta-globin polyadenylation signal. The Δ3egfp cassette has the CAG hybrid promoter, an EGFP missing 81 bp at its 3' end (Δ3egfp) and a rabbit beta-globin polyadenylation signal. This is all followed by a bovine growth hormone polyadenylation sequence.
The entire pRosa26-ΔGF targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus on chromosome 6 via electroporation of 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with 129 females (unspecified substrain) to establish the RaDR-GFP mouse line. Mice were then bred to Gpt-Δ transgenic mice (Tg(gptdelta)1Nmi; unspecified genetic background).

The donating investigator reports that the resulting double mutant RaDR-GFP::Gpt-Δ mice were then backcrossed to C57BL/6J inbred animals for at least ten generations. In 2018, C57BL/6J-congenic RaDR-GFP::Gpt-Δ males with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, they were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation, and the Gpt-Δ transgene was subsequently removed. The resulting B6::RaDR-GFP mice are Stock No. 031413.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-EGFP*)Bpeng}
Name: targeted mutation 1, Bevin Engelward
MGI accession ID: MGI:6114035
Generation method: Targeted
Attributes: Reporter
Synonyms: RaDR-GFP
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: 129S4/SvJae
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Widespread cell types.
Molecular note: The targeting construct contains a PGK-neo-pA cassette followed by a GFP direct repeat HR substrate. Specificly, the PGK-neo-pA cassette containes a single loxP site, the PGK promoter, a neomycin resistance gene and the PGK polyadenylation signal. The green fluorescent protein (GFP) direct repeat homologous recombination (HR) substrate consists of two CAG-DeltaEGFP-polyA expression cassettes arranged in tandem (Delta5egfp :: Delta3egfp). The Delta5egfp cassette has the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an enhanced green fluorescent protein missing 15 bp at its 5' end (Delta5egfp) and a rabbit beta-globin polyadenylation signal. The Delta3egfp cassette has the CAG hybrid promoter, an EGFP missing 81 bp at its 3' end (Delta3egfp) and a rabbit beta-globin polyadenylation signal followed by a bovine growth hormone polyadenylation sequence. The targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.