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.
Bruce M Spiegelman, Dana-Farber Cancer Institute/Harvard Medical School
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).
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 Name||endonuclease-mediated mutation 1, Bruce M. Spiegelman|
|Allele Type||Endonuclease-mediated (Null/Knockout)|
|Gene Symbol and Name||Pm20d1, peptidase M20 domain containing 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.|
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.
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 PM20D1-KO mouse strain in a publication, please cite the originating article(s) and include JAX stock #032193 in your Materials and Methods section.
"MICE" means mouse strains, their progeny derived by inbreeding or crossbreeding, unmodified derivatives from mouse strains or their progeny supplied by The Jackson Laboratory ("JACKSON"). "PRODUCT(S)" means biological materials supplied by JACKSON, and their derivatives. "SERVICES" means projects conducted by JACKSON for other parties that may include but are not limited to the use of MICE or PRODUCTS. "RECIPIENT" means each recipient of MICE, PRODUCTS, or SERVICES provided by JACKSON including each institution, its employees and other researchers under its control. MICE or PRODUCTS shall not be: (i) used for any purpose other than internal research, (ii) sold or otherwise provided to any third party for any use, or (iii) provided to any agent or other third party to provide breeding or other services. Acceptance of MICE, PRODUCTS or SERVICES from JACKSON shall be deemed as agreement by RECIPIENT to these conditions, and departure from these conditions requires JACKSON’s prior written authorization.
MICE, PRODUCTS AND SERVICES ARE PROVIDED "AS IS". JACKSON EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS, IMPLIED, OR STATUTORY, WITH RESPECT TO MICE, PRODUCTS OR SERVICES, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR ANY WARRANTY OF NON-INFRINGEMENT OF ANY PATENT, TRADEMARK, OR OTHER INTELLECTUAL PROPERTY RIGHTS.
In case of dissatisfaction for a valid reason and claimed in writing by a purchaser within ninety (90) days of receipt of, PRODUCTS or SERVICES, JACKSON will, at its option, provide credit or replacement for the PRODUCT received or the SERVICES provided; JACKSON makes no other representations and this shall be the exclusive remedy of the purchaser. Please note specific policy for live mice.
Consistent with the requirement for a written understanding regarding animal care and use, the JACKSON Animal Care and Use Committee will review the animal care and use protocol(s) associated with any SERVICES to be performed at JACKSON, and JACKSON shall have ultimate responsibility and authority for the care of animals while on site or in JACKSON custody.
In no event shall JACKSON, its trustees, directors, officers, employees, and affiliates be liable for any causes of action or damages, including any direct, indirect, special, or consequential damages, arising out of the provision of MICE, PRODUCTS, or SERVICES, including economic damage or injury to property and lost profits, and including any damage arising from acts or negligence on the part of JACKSON, its agents or employees. Unless prohibited by law, in purchasing or receiving MICE, PRODUCTS, or SERVICES from JACKSON, purchaser or recipient, or any party claiming by or through them, expressly releases and discharges JACKSON from all such causes of action or damages, and further agrees to defend and indemnify JACKSON from any costs or damages arising out of any third party claims.
MICE, PRODUCTS or SERVICES are to be used in a safe manner and in accordance with all applicable governmental rules and regulations.
The foregoing represents the General Terms and Conditions applicable to JACKSON’s MICE, PRODUCTS or SERVICES. In addition, special terms and conditions of sale of certain MICE, PRODUCTS, or SERVICES may be set forth separately in JACKSON web pages, catalogs, price lists, contracts, and/or other documents, and these special terms and conditions shall also govern the sale of these MICE, PRODUCTS and SERVICES by JACKSON, and by its licensees and distributors.
Acceptance of delivery of MICE, PRODUCTS or SERVICES shall be deemed agreement to these terms and conditions. No purchase order or other document transmitted by purchaser or recipient that may modify the terms and conditions hereof, shall be in any way binding on JACKSON, and instead the terms and conditions set forth herein, including any special terms and conditions set forth separately, shall govern the sale of MICE, PRODUCTS or SERVICES by JACKSON.