B6.Cg-Cdh6^{tm1.1(cre/ERT2)Jrs}/J

Stock number: 029428
Product name: Cdh6^CreER (Cdh6-CreER)
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

The Cdh6^CreER (Cdh6-CreER) knock-in/knock-out allele was designed to both abolish endogenous gene function and allow the Cdh6 promoter/enhancer regions to direct tamoxifen-inducible Cre recombinase expression to ON-OFF direction-selective retinal ganglion cells (ooDSGCs) that respond to vertical motion (dorsal and ventral), as well as to starburst amacrine cells. These mice may be useful for tamoxifen-inducible recombination of lox-flanked (floxed) alleles in studying photoreceptors and retinal circuitry.

Detailed description

Complex neural circuits underlie mental activities, and defects in circuit assembly may be associated with behavioral disorders. The mouse retina has emerged as an excellent model system for studying neural circuitry. Photoreceptors synapse on bipolar and horizontal interneurons in the outer plexiform layer. Bipolar cells (BCs) carry visual information to the inner plexiform layer (IPL) where the information is processed by amacrine cells and retinal ganglion cells (RGCs); the RGCs carry information to the brain. Cadherin 6 (Cdh6) is a type II cadherin expressed in mouse retina (details below).

The Cdh6^CreER (Cdh6-CreER) knock-in/knock-out allele has a tamoxifen-inducible Cre recombinase gene (CreER^T2 [with N-terminal 6xMyc tag]) inserted into the Cdh6 translational start site - this abolishes endogenous gene function and results in the Cdh6 promoter/enhancer regions directing cre/ERT2 expression.

CreER^T2 fusion gene activity is inducible; observed following tamoxifen administration. When Cdh6^CreER mice are bred with mice containing lox-flanked (floxed) sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in Cdh6-expressing cells in the offspring.


Specifically, tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Cdh6 expression pattern. The donating investigator summarizes that Cdh6^CreER mice have CreER^T2 expression in starburst amacrine cells and selective (though not exclusive) expression in bi-stratified dendrite-retinal ganglion cells (BD-RGCs) - the majority (>90%) of Cdh6-positive RGCs are ON-OFF direction-selective RGCs (ooDSGCs) that respond to vertical motion (dorsal and ventral). Cdh6-positive ooDSGCs comprise ~8% of all RGCs.

The donating investigator has not observed Cre recombinase activity prior to tamoxifen exposure. Although no expression of CDH6 RNA (by qPCR) is reported from this knock-in/knock-out allele, both homozygotes (Cdh6^CreER/CreER) and heterozygotes (Cdh6^CreER/+) are viable and fertile with no reported phenotype abnormalities or RGC defects to date (August 2016).

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

The Cdh6^CreER (Cdh6-CreER) knock-in/knock-out allele was created in the laboratory of Dr. Joshua R. Sanes (Harvard University). The targeting vector contained (from 5' to 3') a cDNA sequence encoding the CreER^T2 fusion gene (Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain) with N-terminal 6xMyc tag, followed by a frt-flanked SV40-neo selection cassette. This was inserted into the translational start site in the first coding exon of the cadherin 6 locus (Cdh6) on chromosome 15 via electroporation of (C57BL/6 x 129S4/SvJae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6J for germline transmission. At some point, they were bred with Tg(ACTFLPe)9205Dym mice (on a C57BL/6J genetic background; Stock No. 005703) for germline deletion of the SV40-neo selection cassette. The resulting Cdh6^CreER (Cdh6-CreER) colony was backcrossed to C57BL/6J wildtype mice for at least ten generations (and the Flp-expressing transgene was removed). The donating investigator reports some breeding with C57BL/6J-congenic floxed/fluorescent strains have also occurred, but the Cdh6^CreER mice no longer have those floxed/fluorescent alleles. In 2016, Cdh6^CreER males with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
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).

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 rederived living colony at The Jackson Laboratory Repository. Three of the 27 markers throughout the genome were segregating, suggesting an incomplete backcross. One of 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/6J ; C57BL/6N genetic background.

Allele

Symbol: Cdh6^{tm1.1(cre/ERT2)Jrs}
Name: targeted mutation 1.1, Joshua R Sanes
MGI accession ID: MGI:5461051
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible; Null/Knockout
Synonyms: Cdh6^{tm1(cre/ERT)Jrs}; Cdh6-CreER
Marker symbol: Cdh6
Marker name: cadherin 6
Marker synonyms: cad6; KCAD; K-cadherin
Chromosome: 15
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: When induced, Cre recombinase activity recapitulates the endogenous Cdh6 expression pattern in starburst amacrine cells and selective expression in bi-stratified dendrite-retinal ganglion cells.
Molecular note: The targeting vector contained (from 5' to 3') a cDNA sequence encoding the CreER fusion gene (Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain) with N-terminal 6xMyc tag, followed by an FRT-flanked SV40-neo selection cassette. This was inserted into the translational start site in the first coding exon of the gene. Flp-mediated recombination removed the FRT-flanked neo cassette. Mice were crossed to reporter animals for analysis.