Homozygous γ-4- knockout mice exhibit dysfunctional synaptic targeting of AMPARs. These mice may be useful in studying AMPAR membrane trafficking and synaptic transmission/targeting in the brain; specifically in the olfactory bulb, striatum and glia of developing brain.
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-4 is expressed transiently throughout the developing brain, with especially high levels in the striatum. It is the primary TARP expressed in the olfactory bulb, striatum and glia, and may be the sole TARP expressed in neonates. γ-4 regulates AMPAR abundance and kinetics.
The TARP γ-4 knockout allele (γ-4-) has a neomycin resistance gene replacing the exon encoding the first extracellular domain and the second and third transmembrane domains. Homozygous mice (γ-4-/- or Cacng4-/-) exhibit dysfunctional synaptic targeting of AMPARs (abnormal excitatory postsynaptic currents). However, homozygotes show no obvious difference in GluR1 and GluR2/3 expression when compared to wildtype controls. Homozygous mice are viable and fertile. Heterozygous mice are viable and fertile with no reported abnormalities. Western blot analysis of brain protein extracts confirms the absence of protein expression from the knockout allele.
The γ-4- 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 exon 3 (encoding first extracellular domain and the second and third transmembrane domains) of the TARP γ-4 locus (Cacng4) on chromosome 11. 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-4- mice were bred together for several generations. The donating investigator reports that homozygous males (black coat color) on a C57BL/6 genetic background were sent to The Jackson Laboratory Repository in 2017 (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).
In 2017, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 first generation rederived living colony at The Jackson Laboratory Repository. This revealed markers on a total of 6 chromosomes that were not fixed for C57BL/6 allele-type (i.e., still segregating for 129S allele-type markers). In addition, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (note the allele-type for C57BL/6N and 129S are the same for those 2 markers). Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were previously bred to an unspecified mouse (129S contributions), and the C57BL/6 genetic background contributions may be a mix of C57BL/6J;C57BL/6N.
|Allele Name||targeted mutation 1, Roger A Nicoll|
|Allele Type||Targeted (Null/Knockout)|
|Allele Synonym(s)||gamma 4; Tarp gamma 4|
|Gene Symbol and Name||Cacng4, calcium channel, voltage-dependent, gamma subunit 4|
|Strain of Origin||C57BL/6|
|Molecular Note||Exon 3 was replaced with a neomycin resistance cassette. Western blot analysis confirmed the absence of protein product.|
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 γ-4- mouse strain in a publication, please cite the originating article(s) and include JAX stock #028445 in your Materials and Methods section.