C57BL/6J-Pm20d1^em1Brsp/J

Stock number: 032193
Product name: PM20D1-KO
Research areas: Cardiovascular Research, Diabetes and Obesity Research, Metabolism Research, Research Tools, Sensorineural Research

Description

PM20D1-KO is a CRISPR/cas9-made knock-out allele with a Δ6-bp out-of-frame deletion in Pm20d1 exon 1 that generates an early stop codon. These mice may be useful in studying PM20D1 function in the bidirectional regulation of tissue and circulating N-acyl amino acids (NAAs), as well as NAA physiology in glucose homeostasis, energy metabolism and pain sensation.

Detailed description

N-acyl amino acids (NAAs) are bioactive signaling lipids with pleiotropic physiologic functions. PM20D1 has a dominant role in the bidirectional regulation of both tissue NAAs and circulating NAAs.

PM20D1-KO is a CRISPR/cas9-made knock-out allele with a Δ6-bp out-of-frame deletion in Pm20d1 exon 1 that generates an early stop codon. While mice homozygous for this global knock-out allele (PM20D1-KO) are viable and fertile with no reported gross phenotypic or behavioral abnormalities, they exhibit a variety of metabolic and pain phenotypes - including impaired glucose tolerance and insulin resistance in response to higher fat diet, elevated body temperature in response to cold stimuli, and anti-nociceptive behaviors. More details are described below.

PM20D1-KO tissues show PM20D1 protein levels are completely abolished. Homozygotes show reduced NAA synthase/hydrolase activity. Specifically, multiple PM20D1-KO tissues show consistently elevated levels of the metabolite N-oleoyl-glutamine (C18:1-Gln), an antagonist of transient receptor potential nociceptor channel V1 (TRPV1).

On standard chow diet, PM20D1-KO mice were indistinguishable from wildtype in several metabolic parameters (body weight, energy expenditure, food intake, glucose homeostasis). When maintained on high fat diet (HFD ; 60% kcal from fat) or Western diet (WD ; 40% kcal from fat, 0.2% cholesterol), homozygotes show no major effects on body weight or adiposity compared to similarly-treated wildtype mice. However, PM20D1-KO mice exhibit impaired glucose tolerance after 16 weeks HFD or 17 weeks WD, as well as reduced insulin sensitivity after 19 weeks WD.

In response to environmental cold challenge, PM20D1-KO mice remained significantly warmer than control mice.

Compared to similarly-treated wildtype animals, PM20D1-KO mice exhibit antinociceptive behaviors selectively in response to chemical and inflammatory pain stimuli, while maintaining normal thermal pain sensation and normal movement/coordination.

The phenotype of mice heterozygous for the PM20D1-KO allele has not been characterized to date (June 2018).

Development

The global PM20D1-KO allele was created in the laboratory of Dr. Bruce M. Spiegelman (Dana-Farber Cancer Institute/Harvard Medical School) using CRISPR/Cas9 genome engineering.
First, two guide RNAs were designed to target sequences in exon 1 of the peptidase M20 domain containing 1 locus (Pm20d1) on chromosome 1. The guide RNAs and a CRISPR/cas9-expressing plasmid were injected into C57BL/6J embryos. Founders were screened by PCR and sequencing, and founder female 1754#4 was identified with exon 1 containing an out-of-frame deletion of 6 basepairs (Δ6-bp) that results in the conversion of a phenylalanine codon into a premature stop codon (TTC->TAG). Specifically, Sanger sequencing confirmed the presence of a new T-A junction and a new stop codon in exon 1 at the 43rd amino acid position. qPCR analysis of this PM20D1-KO allele confirmed the Δ6-bp modification was also present in its mRNA. Shotgun proteomics confirmed selective loss of PM20D1 protein from livers of PM20D1-KO mice.
This founder was bred to C57BL/6J male mice for germline transmission. The PM20D1-KO colony was backcrossed to C57BL/6J for a total of two generations, and then bred together as heterozygotes. The mice have not been characterized for off-target mutations to date (June 2018).
In 2018, 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).

Allele

Symbol: Pm20d1^em1Brsp
Name: endonuclease-mediated mutation 1, Bruce M. Spiegelman
MGI accession ID: MGI:6162684
Generation method: Endonuclease-mediated
Attributes: Null/Knockout
Synonyms: PM20D1-KO
Marker symbol: Pm20d1
Marker name: peptidase M20 domain containing 1
Marker synonyms: 4732466D17Rik; Cps1; RGD1564063
Chromosome: 1
Strain of origin: C57BL/6J
Molecular note: CRISPR/Cas9 targeting is used to introduce an out-of-frame deletion of 6 basepairs in exon 1 that results in the conversion of a phenylalanine codon into a premature stop codon (TTC-->TAG). Sanger sequencing confirmed the presence of a new T-A junction and a new stop codon in exon 1 at the 43rd amino acid position. qPCR analysis confirmed the modification was present in its mRNA. Shotgun proteomics confirmed selective loss of PM20D1 protein from liver.