The RC::L-DTA allele has a cre-dependent FLEx switch containing an eGFP sequence and an inverted tox176 attenuated diphtheria toxin subunit alpha gene (DTA*G128D) that are uniquely flanked/separated by inward-facing lox sites (both lox2272 and loxP). Widespread eGFP fluorescence is observed in the absence of Cre recombinase. Subsequent exposure to Cre recombinase that places the DTA*G128D into the proper orientation for expression results in ablation of those cells.
Patricia Jensen, National Institute of Environmental Health Sciences (NIEHS)
Genetic Background | Generation |
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N4+N1F4
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Allele Type | Gene Symbol | Gene Name |
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Targeted (Conditional ready (e.g. floxed), Reporter, Inserted expressed sequence) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
Starting at:
$255.00 Domestic price for female 4-week |
333.51 Domestic price for breeder pair |
The RC::L-DTA allele is designed with a cre-dependent FLEx switch containing an eGFP sequence and an inverted tox176 attenuated diphtheria toxin subunit alpha gene (DTA*G128D) that are uniquely flanked/separated by inward-facing lox sites (both lox2272 and loxP). Under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread eGFP fluorescence is observed in the absence of Cre recombinase. Subsequent exposure to Cre recombinase that places DTA*G128D into the proper orientation to be expressed results in ablation of the cre-expressing cells. RC::L-DTA mice allow selective ablation in a tissue/cell-specific manner. The RC::L-DTA allele is uniquely suitable to abrogate any unwanted, ectopic cell death resulting from the intrinsic leakiness of the transcriptional regulatory sequences in the experimental model before Cre recombinase, and also to avoid the extreme toxicity of diphtheria toxin to affect any undesired animal cell populations after Cre recombinase.
Specifically, the donating investigator reports that in the absence of Cre recombinase, mice appear phenotypically normal with robust and ubiquitous eGFP expression when visualized by direct fluorescence in embryos and adult brain. The donating investigator reports that homozygous mice are viable with no gross abnormalities. Although homozygous females are fertile, homozygous males appear to be infertile (unknown cause), and heterozygous males have been very sub-fertile.
Several cre-mediated recombination outcomes of the RC::L-DTA allele are possible based on which lox sites are recombined. The outcomes are described below, along with some experimental considerations:
i. inversion of the RC::L-DTA allele at the lox2272 sites places the DTA*G128D sequence adjacent to the CAG promoter and into the proper orientation for expression; resulting in ablation of the cre-expressing cell type.
ii. inversion of the RC::L-DTA allele at the loxP sites places the DTA*G128D into the proper orientation for expression, but the eGFP cassette (now flanked with lox2272 sites in the same orientation) remains adjacent to the CAG promoter; resulting in continued eGFP expression and no deletion of the cre-expressing cell type.
iii. recombination of outcome ii at the lox2272 sites now deletes the eGFP cassette, placing the DTA*G128D sequence adjacent to the CAG promoter and into the proper orientation for expression; resulting in ablation of the cre-expressing cell type.
iv. inversion of outcome ii at the loxP sites reverts to the original RC::L-DTA allele; resulting in continued eGFP expression and no deletion of the cre-expressing cell type.
The two lox variants, lox2272 and loxP, are compatible only with a lox sequence identical to self; they do not recombine with each other. Although some RC::L-DTA recombination outcomes require more than one Cre recombinase pulse to achieve DTA expression, no outcomes result in deletion of the DTA cassette. Because inward-facing lox sequences result in cre-mediated inversion rather than excision, surviving cells may be able to reversibly switch back-and-forth from the non-DTA-expressing orientations as long as Cre recombinase is active in the cell. Those surviving cells (outcome ii and the original RC::L-DTA allele) may eventually recombine to a DTA-expressing orientation that results in cell death. To this point the donating investigator reports that, regardless of which inversion event occurs first, continuous exposure to Cre rapidly recombines the allele into the final state (DTA in correct orientation for expression).
The tox 176 attenuated diphtheria toxin A chain (DTA*G128D) exhibits diminished enzyme activity/toxicity compared to diphtheria toxin, but retains substantial toxicity levels for ablation of mammalian cells. RC::L-DTA has this attenuated DTA in a transcriptionally-inverted confirmation located downstream of an EGFP-polyA sequence prior to Cre recombinase. This makes RC::L-DTA mice uniquely suitable to avoid the extreme toxicity of diphtheria toxin to animal cells and abrogate any unwanted, ectopic cell death resulting from the intrinsic leakiness of the transcriptional regulatory sequences in the experimental model.
The RC::L-DTA allele has a CAG hybrid promoter followed by a variant lox-flanked region containing an eGFP cassette and an inverted attenuated diptheria toxin subunit alpha gene (DTA*G128D), all inserted into the Gt(ROSA)26Sor locus. The specific details are below.
The pRosa-CAG-rox-FRT-lox-eGFP-DTA targeting vector (pR/C-RFLGD) was created in the laboratory of Dr. Patricia Jensen (National Institute of Environmental Health Sciences). The vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a rox-flanked His3-SV40 transcriptional STOP cassette, a frt-flanked His3-SV40 transcriptional STOP cassette, a cre-dependent FLEx switch (containing [from 5' to 3'] an inward-facing lox2272 site, a cDNA sequence encoding enhanced green fluorescent protein [eGFP], a rabbit β-globin polyA signal, an inward-facing loxP site, a reverse complimentary sequence encoding the tox 176 attenuated diphtheria toxin A chain [G-to-A transition at nt 383 encodes glycine-to-aspartic acid at amino acid 128 (DTA*G128D)], a second inward-facing lox2272 site, a second inward-facing loxP site ), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, and an attB/attP-flanked PGK-frt-Neo-polyA cassette. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. ES cells were then transiently transfected with a PhiC31-expressing plasmid to remove the attB/attP-flanked PGK-frt-Neo-polyA cassette and replace it with the recombined attB/attP site (attL). Following ES cell injection into recipient blastocysts, chimeric mice with the RC::RFL-DTA allele were bred to C57BL/6J mice for germline transmission. The RC::RFL-DTA colony was backcrossed with C57BL/6J wildtype mice for at least 12 generations. Next, they were then crossed to mice expressing germline Dre recombinase [Tg(CAG-dre)1Afst (N19 onto C57BL/6J)] and then to mice expressing germline Flp recombinase [B6.Cg-Tg(ACTFlpe)9205Dym/J (Stock No. 005703)]. The resulting offspring with the RC::L-DTA genotype (deletion of both the rox-flanked STOP and frt-flanked STOP sequences) were identified. The RC::L-DTA colony was bred with C57BL/6J wildtype mice for at least four generations. In 2019, RC::L-DTA mice (segregating for Tg(ACTFlpe)9205Dym) 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 Tg(ACTFlpe)9205Dym was removed.
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).
Expressed Gene | Dta, Diphtheria toxin A chain, |
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Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | EGFP fluorescence is widespread in the absence of Cre recombinase. Subsequent exposure to Cre recombinase allows expression of attenuated diphtheria toxin (DTA*G128D) resulting in ablation of those cre expressing cells. |
Allele Name | targeted mutation 2.1, Patricia Jensen |
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Allele Type | Targeted (Conditional ready (e.g. floxed), Reporter, Inserted expressed sequence) |
Allele Synonym(s) | RC::L-DTA |
Gene Symbol and Name | Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano |
Gene Synonym(s) | |
Promoter | CAG, CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, |
Expressed Gene | Dta, Diphtheria toxin A chain, |
Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | EGFP fluorescence is widespread in the absence of Cre recombinase. Subsequent exposure to Cre recombinase allows expression of attenuated diphtheria toxin (DTA*G128D) resulting in ablation of those cre expressing cells. |
Strain of Origin | (129S6/SvEvTac x C57BL/6NCrl)F1 |
Chromosome | 6 |
Molecular Note | The targeting vector iss designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a rox-flanked His3-SV40 transcriptional STOP cassette, a frt-flanked His3-SV40 transcriptional STOP cassette, a cre-dependent FLEx switch (containing [from 5' to 3'] an inward-facing lox2272 site, a cDNA sequence encoding enhanced green fluorescent protein [eGFP], a rabbit -globin polyA signal, an inward-facing loxP site, a reverse complimentary sequence encoding the tox 176 attenuated diphtheria toxin A chain [G-to-A transition at nt 383 encodes glycine-to-aspartic acid at amino acid 128 (DTA*G128D)], a second inward-facing lox2272 site, a second inward-facing loxP site ), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, and an attB/attP-flanked PGK-frt-Neo-polyA cassette. This entire targeting vector is inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. A PhiC31-expressing plasmid is used to remove the attB/attP-flanked PGK-FRT-Neo-polyA cassette and replace it with the recombined attB/attP site (attL). Flp-mediated recombination removed the FRT-flanked STOP sequence. Dre-mediated recombination removed the rox-flanked STOP sequence. The resulting allele has a CAG hybrid promoter followed by a variant lox-flanked region containing an eGFP cassette and an inverted attenuated diptheria toxin subunit alpha gene (DTA*G128D). GFP expression is visualized by direct fluorescence in embryos and adult brain. |
When maintaining a live colony, heterozygous females may be bred to wildtype males from the colony, or to C57BL/6J inbred males (Stock No. 000664). The donating investigator reports that homozygous males appear to be infertile (unknown cause), and heterozygous males have been very sub-fertile.
When using the RC::L-DTA mouse strain in a publication, please cite the originating article(s) and include JAX stock #026944 in your Materials and Methods section.
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