The Arf6Flox allele has loxP sites flanking the entire coding region of the ADP-ribosylation factor 6 gene. Removal of the floxed sequences creates a null allele. These mice may be useful in studying protein trafficking/vesicular transport, lipid signaling, cancer and neurodegeneration, as well as intracellular cholesterol distribution via regulation of the retromer pathway.
Gilbert Di Paolo, Columbia University Medical Center
ADP-ribosylation factor 6 (ARF6) is a GTP-binding protein involved in various aspects of membrane dynamics and cell physiology. ARF6 functions primarily as a regulator of protein trafficking/vesicular transport to and from the plasma membrane, as well as endocytic recycling, cytoskeleton remodeling, lipid signaling and phosphoinositide signaling. ARF6 is also involved in the control of intracellular cholesterol distribution.
The Arf6Flox allele has loxP sites flanking the entire coding region of the Arf6 gene. Mice homozygous for Arf6Flox are viable and fertile with no reported abnormalities. Following exposure to Cre recombinase, the floxed sequences are deleted in the cre-expressing tissues; creating a null allele. These Arf6Flox may be bred to Cre recombinase mice to generate tissue or cell type-specific conditional knockout mice.
For example, breeding Arf6Flox mice to germline cre-expressing mice results in the ARF6 global knockout allele, which is embryonic lethal when homozygous.
In addition, Arf6Flox animals bred to C57BL/6 ROSACre-ER mice (Stock No. 008463) can be used to create a tamoxifen-inducible knockout model of mouse embryonic fibroblasts (MEFs).
The Arf6Flox allele was created by Dr. Gilbert Di Paolo (Columbia University Medical Center). A targeting vector was designed to have a loxP site upstream, and a frt-flanked neo cassette with 3' loxP site downstream of the entire coding region (~4000 bp) of the ADP-ribosylation factor 6 gene (Arf6) on chromosome 12. The construct was electroporated into C57BL/6 x 129/SvJ embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6J females to establish the Arf6Flox Neo colony. Mice were then bred to the C57BL/6J-congenic FLP deleter strain (Stock No. 005703) for germline removal of the frt-flanked neo. The donating investigator reported that the Arf6Flox colony was backcrossed to C57BL/6J wildtype mice for at least seven generations (and the Flp-expressing transgene was removed) prior to sending males with black coat color The Jackson Laboratory Repository in 2016 (see SNP results below).
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/6 inbred male (thus the Y chromosome of the congenic strain is of C57BL/6 origin).
In 2016, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. This revealed only 1 of 27 markers not fixed for C57BL/6 allele-type (i.e., a marker on the same chromosome as the Arf6 locus was still segregating for 129S allele-type). In addition, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (chromosomes 11 [~4.4 Mbp] and 19 [~49.9 Mbp]; note the allele-type for C57BL/6N and 129S are the same for these 5 markers). Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background.
|Allele Name||targeted mutation 1.1, Gilbert Di Paolo|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Gene Symbol and Name||Arf6, ADP-ribosylation factor 6|
|Strain of Origin||C57BL/6 x 129/Sv|
|Molecular Note||A targeting vector was designed to have a loxP site upstream, and a FRT-flanked neo cassette with 3' loxP site downstream of the entire coding region (~4000 bp) of the gene. Flp-mediated recombination removed the FRT-flanked neo cassette.|
Mice homozygous for Arf6Flox are viable and fertile with no reported abnormalities. When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
When using the Arf6Flox mouse strain in a publication, please cite the originating article(s) and include JAX stock #028669 in your Materials and Methods section.