B6;129-Slc15a2^tm1Dsmi/JrsJ

Stock number: 028766
Product name: PEPT2 knock-out
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

These PEPT2 knock-out (lacZ knock-in) mice exhibit an enhanced analgesic response to L-kyotorphin and may be useful in studies of oligopeptide transport.

Detailed description

The oligopeptide-proton symporter that is encoded by the Slc15a2 gene facilitates movement of small peptides and peptidomimetic drugs across the cell membrane. These PEPT2 deficient mice carry a knock-out allele of the Slc15a2 gene in which a lacZ/NEO cassette replaced part of exon 1 and exons 2 & 3, which encodes the first 112 amino acids.

Mice that are homozygous for the targeted mutation are viable and fertile. No gene product (mRNA or protein) is detected in homozygotes by Northern blot analysis of kidney tissue, RT-PCR analysis of choroid plexus, and Western blot analysis of kidney apical membrane vesicles.

Clearance of the endogenous analgesic L-kyotorphin neuropeptide from cerebrospinal fluid (CSF) is slower in homozygotes compared to wildtype controls. Homozygotes exhibit an enhanced analgesic response (5.2-fold more potent antinociceptive response) to L-kyotorphin compared to wildtype mice. After exogenous dosing of carnosine in homozygotes, kidney uptake of the dipeptide carnosine is increased and renal clearance decreased. Endogenous carnosine levels are reduced in homozgyotes. Primary cultured astrocytes from homozygotes exhibit about a 50% reduction in uptake of L-kyotorphin compared to wildtype astrocytes.

The gap junction-permeable fluorophore-coupled dipeptide, beta-alanine-lysine-N-7-amino-4-methylcoumarin-3-acid (βALA) is not taken up by retinal Muller glia of homozygotes.

Development

A targeting vector designed by Dr. David E. Smith (University of Michigan) containing a lacZ/NEO cassette was used to disrupt part of exon 1, exons 2 and 3. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺ derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric male animals were crossed to C57BL/6J female mice. Heterozygotes were intercrossed to generate homozygotes. Dr. Joshua Sanes (Harvard University) obtained the knock-out mice from Dr. David E. Smith, and then backcrossed the mice to C57BL/6J once. 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: Slc15a2^tm1Dsmi
Name: targeted mutation 1, David E Smith
MGI accession ID: MGI:2451265
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Slc15a2
Marker name: solute carrier family 15 (H+/peptide transporter), member 2
Chromosome: 16
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: lacZ,beta-galactosidase,E. coli
Molecular note: A fragment encompassing a portion of exon 1 and all of exons 2 and 3 was replaced by a lacZ-neo cassette inserted by homologous recombination. The deleted region encoded the first 112 amino acids. Transcript was undetected in homozygous mutant mice by Northern blot analysis of renal RNA and RT-PCR analysis of choroid plexus RNA. Western blot analysis indicated an absence of encoded protein in homozygous mutant kidney brush border vesicle preparations.