c-Kitlox66-71 is a Cre-recombinase conditional knockout/eGFP-reporter allele - allowing cre-inducible c-Kit deletion and concomitant labeling of the c-Kit-deficient cells with eGFP. These mice are useful for imaging of c-Kit-deficient cells in vivo as well as facilitating isolation of these cells in vitro.
Shahin Rafii, Weill Cornell Medical College
Genetic Background | Generation |
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Allele Type | Gene Symbol | Gene Name |
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Targeted (Reporter, Null/Knockout) | Kit | KIT proto-oncogene receptor tyrosine kinase |
c-Kitlox66-71 is a Cre-recombinase conditional knockout/eGFP-reporter allele - allowing cre-inducible c-Kit deletion and concomitant labeling of the c-Kit-deficient cells with eGFP.
Prior to Cre recombination, the c-Kitlox66-71 allele will transcribe a full-length c-Kit transcript, and no eGFP expression is reported.
Exposure to Cre recombinase will invert the fragment bordered by the inward-facing lox71 and lox66 sites (lox71-SA-IRES-eGFP-2pA-lox66) into sense orientation; creating the eGFP-expressing c-Kit knockout allele (c-KitΔGFP). Specifically, the transcriptional stops upstream of the exons encoding the transmembrane and kinase domains of c-Kit result in a truncated protein and loss of c-Kit cell surface expression. The splice-acceptor-IRES sequences lead to eGFP expression in c-Kit-expressing cells (direct fluorescence). The donating investigator reports eGFP direct fluorescence is observable by flow cytometry, but weak by imaging. These mice are useful for imaging of c-Kit-deficient cells in vivo as well as facilitating isolation of these cells in vitro.
The c-Kit lox66-71 allele was designed by Dr. Shahin Rafii (Weill Cornell Medical College). A targeting vector was designed to insert the following, all in reverse transcriptional orientation, into intron 8 of the kit oncogene locus (Kit; c-Kit): frt-flanked PGK-neo-pA cassette, inward-facing lox71 site, splice-acceptor, IRES-eGFP-2pA, inward-facing lox66 site. C57BL/6-
Tyrc-2J-derived C2J ES cells. Correctly targeted ES cells were transiently transfected with a Flippase-puromycin plasmid to remove the PGK-neo-pA cassette. Neo-excised ES cells were injected into recipient blastocysts and chimeric males were bred with C57BL/6-Tyrc-2J/J female mice. The donating investigator reports that the c-Kitlox66-71 mice were crossed to C57BL/6; the albino allele (Tyrc-2J)has been bred out.
Expressed Gene | GFP, Green Fluorescent Protein, |
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Site of Expression | EGFP is expressed in c-Kit-expressing cells. |
Allele Name | targeted mutation 1.1, Shahin Rafii |
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Allele Type | Targeted (Reporter, Null/Knockout) |
Allele Synonym(s) | c-Kit lox66-71 |
Gene Symbol and Name | Kit, KIT proto-oncogene receptor tyrosine kinase |
Gene Synonym(s) | |
Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | EGFP is expressed in c-Kit-expressing cells. |
Strain of Origin | B6(Cg)-Tyrc-2J/J |
Chromosome | 5 |
Molecular Note | A construct containing a lox66 site, inverted GFP gene, lox71, and FRT-flanked neo cassette was inserted between exon 8 and exon 9. Flp-mediated recombination removed the neo cassette. |
While maintaining a live colony, these mice are bred as heterozygotes. The donating investigator has not attempted to breed homozygous mice. (July 2016)
When using the c-Kitlox66-71 (c-Kit lox66-71) mouse strain in a publication, please cite the originating article(s) and include MMRRC stock #42035 in your Materials and Methods section.
Facility Barrier Level Descriptions
The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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