Homozygous γ-8- knockout mice exhibit dysfunctional AMPAR expression, impaired synaptic plasticity and dysfunctional maternal behavior. These mice may be useful in studying AMPAR membrane trafficking and synaptic transmission/targeting in the brain; specifically in the hippocampus.
Roger A Nicoll, University of California, San Francisco
Synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are the dominant glutamate receptor in the brain. AMPARs are regulated by a family of auxiliary subunits known as transmembrane AMPAR regulatory proteins (TARPs) and the additional AMPAR auxiliary subunits/binding proteins (cornichon).
TARP subtypes are the prototypical stargazin (γ-2), and the homologous γ-3, γ-4 (Cacng4), and γ-8 (Cacng8). The TARP subtypes are differentially expressed throughout the CNS; this imparts functional diversity to AMPARs in distinct neuronal populations. Specifically, γ-8 is the primary TARP expressed in hippocampus.
The TARP γ-8 knockout allele (γ-8-) has a neomycin resistance gene replacing two exons encoding the transmembrane domain. Homozygous mice (γ-8-/- or Cacng8-/-) exhibit dysfunctional AMPAR expression and impaired synaptic plasticity (reduced LTP). Specifically, homozygotes show a substantial loss and mislocalization of hippocampal GluR1 and GluR2/3 proteins. γ8-/- exhibit reduced hippocampal synaptic AMPAR currents, extrasynaptic receptor depletion, synaptic pool impairments and long term potentiation reduction.
Homozygotes are viable and fertile, born in Mendelian ratios, and have no gross behavioral or anatomical defects. The donating investigator reports that homozygous females have dysfunctional maternal behavior. Heterozygous mice are viable and fertile with no reported abnormalities. Quantitative immunoblot of hippocampal extracts confirms the absence of protein expression from the knockout allele.
The γ-8- knockout allele was created by Dr. Roger A. Nicoll (University of California, San Francisco). A targeting vector was designed to have neomycin resistance gene replacing two exons encoding the transmembrane domain (exons 2-3) of the TARP γ-8 locus (Cacng8) on chromosome 7. The construct was electroporated into C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with C57BL/6J for germline transmission. The resulting γ-8- mice were bred together for several generations and/or backcrossed to C57BL/6J mice for several generations, and then males with black coat color were sent to The Jackson Laboratory Repository in 2016 (see SNP notes 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 SNP (single nucleotide polymorphism) panel analysis, with 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 two males sent to The Jackson Laboratory Repository. This revealed each mouse had a single marker that was not fixed for C57BL/6 allele-type (one on chromosome 1 [~87 Mbp], the other on chromosome 12 [~30 Mbp]). In addition, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice. Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were a mix of C57BL/6J and C57BL/6N, and also had an undetermined genetic background contribution.
In 2017, a 48 SNP panel was performed on our first generation rederived living colony. This revealed 93-98% of the markers tested were C57BL/6 allele-type, with up to 3 markers not fixed for C57BL/6 allele-type (one each on chromosomes 5 [~108 Mbp], 7 [~36 Mbp] and 12 [~30 Mbp]). All five C57BL/6 substrain markers tested as C57BL/6J allele-type.
|Allele Name||targeted mutation 1, Roger A Nicoll|
|Allele Type||Targeted (Null/Knockout)|
|Gene Symbol and Name||Cacng8, calcium channel, voltage-dependent, gamma subunit 8|
|Strain of Origin||C57BL/6|
|Molecular Note||The locus was disrupted by the replacement of exons 2 and 3 with a neomycin resistance gene. Quantitative immunoblot of hippocampal extracts of mutants confirms the absence of protein.|
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, heterozygous females may be bred to homozygous males. The donating investigator reports that homozygous females have dysfunctional maternal behavior.
When using the γ-8- mouse strain in a publication, please cite the originating article(s) and include JAX stock #028446 in your Materials and Methods section.