STOCK Pld1^tm1.1Gdp/J

Stock number: 028665
Product name: Pld1^-
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Metabolism Research, Neurobiology Research, Research Tools

Description

The Pld1^- null allele has exons 13-14 (including the catalytic activity-dependent first HKD motif) of the phospholipase D1 gene deleted. These mice may be useful in studying membrane trafficking, membrane fusion, cancer and neurodegeneration.

Detailed description

Phospholipase D1 (Pld1) and its related isoform phospholipase D2 (Pld2) hydrolyze phosphatidylcholine to generate bioactive lipid phosphatidic acid (PA), are implicated in membrane trafficking/membrane fusion, and are elevated/up-regulated in various human carcinomas (including colorectal cancer).


The Pld1^- null allele has exons 13-14 (including the catalytic activity-dependent first HKD motif) of the phospholipase D1 gene deleted. Homozygous mice are viable and fertile with no reported abnormalities. No protein expression from the knockout allele is detected by western blot analysis of homozygous liver, brain and embryonic fibroblasts.

To study the effect of PLD1-deficiency on spontaneous intestinal tumorigenesis, the Pld1^- animals can be bred to Apc^Min mice (Stock No. 002020). Specifically, on a C57BL/6J genetic background, PLD1-deficient Apc^Min/+ mice have significantly reduced intestinal tumorigenesis and increased survival. The protection is less significant when Apc^Min/+ mice are heterozygous for Pld1^-. Similar PLD1-deficiency induced protection was observed for an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model.
In contrast, deficiency of the related isoform PLD2 (Stock No. 028668) does not significantly reduce intestinal tumorigenesis in the C57BL/6J-Apc^Min model.

Development

The Pld1^- null allele was created by Dr. Gilbert Di Paolo (Columbia University Medical Center). A targeting vector was designed to have a loxP site upstream of exon 13, and a frt-flanked neo cassette with 3' loxP site just downstream of exon 14 of the phospholipase D1 gene (Pld1) on chromosome 3. 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 Pld1^{Flox Neo} colony. The mice were then bred to the C57BL/6J-congenic FLP deleter strain (Stock No. 005703) for germline removal of the frt-flanked neo. The resulting Pld1^Flox colony was backcrossed to C57BL/6J wildtype mice for several generations (and the Flp-expressing transgene was removed). The donating investigator reports that mice were bred with the C57BL/6-congenic Cre deleter strain (Stock No. 003724) for germline deletion of the floxed sequences (exons 13-14 and single frt site); creating the Pld1^- null allele. The donating investigator reported the Pld1^- colony had been backcrossed to C57BL/6J a total of eight generations (and the cre-expressing transgene was removed) prior to sending males with black coat color to 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/6J inbred male (thus the Y chromosome of the congenic strain should be of C57BL/6J 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 mice sent to The Jackson Laboratory Repository, as well as a cohort of our first generation rederived mice. In both assays, this showed 2 of 27 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for 129 allele-type markers): one on chromosome 3 (~10 Mbp away from Pld1 locus) and one on chromosome 13. In addition, 1 of 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating (chromosome 13) - 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 mostly C57BL/6J, and they retained a region of chromosome 13 that is derived from non-C57BL/6 origin.

Allele

Symbol: Pld1^tm1.1Gdp
Name: targeted mutation 1.1, Gilbert Di Paolo
MGI accession ID: MGI:5556064
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Pld1
Marker name: phospholipase D1
Chromosome: 3
Strain of origin: C57BL/6 x 129/SvJ
Molecular note: A loxP site was inserted upstream of exon 13. An FRT-flanked neomycin resistance cassette with a 3' loxP site was inserted downstream of exon 14. This allele is created by using either Cre-mediated recombination to remove exon 13, exon 14 and the selection cassette or Flp-mediated recombination to remove the FRT-flanked neo cassette, followed by Cre-mediated recombination to remove exons 13 and 14 . Western blot analysis confirmed the absence of protein expression in the liver and mouse embryonic fibroblasts.