C57BL/6J-Gt(ROSA)26Sor^{em1(CAG-PACS1*R203W)Gath}/J

Stock number: 038551
Product name: R26^P1R203W , ROSA26-LSL-PACS1-R203W , Gt(ROSA)26Sor^{tm1(CAG-LSL-Pacs1-R203W)Gath}
Research areas: Developmental Biology Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

R26^P1R203W knock-in mice have conditional, widespread expression of the human PACS1 (phosphofurin acidic cluster sorting protein 1) gene with the human R203W variant associated with with developmental delay and intellectual disability (PACS1 syndrome). This strain may be useful for studies involving research in neurodevelopmental disorders such as PACS1 syndrome.

Of note, the wild-type human PACS1 gene is distributed as C57BL/6J-Gt(ROSA)26Sor^{em2(CAG-PACS1)Gath}/J (Stock No. 038552). In addition, the orthologous mouse R201W mutation knocked in to the mouse Pacs1 locus is distributed as C57BL/6J-Pacs1^em2Gath/J (Stock No. 038550).

Detailed description

The Pacs1 (phosphofurin acidic cluster sorting protein 1) gene encodes a trafficking protein involved in trans-Golgi network membrane protein localization.  A human missense mutation in PACS1, R203W, is associated with developmental delay and intellectual disability (PACS1 syndrome). Functionally, the mutation results in increased dendrite arborization, diminished spine density, and fewer functional synapses.

R26^P1R203W conditional knockin mice carry a CAG promoter followed by a floxed STOP, and the hemagluttinin (HA)-tagged human PACS1 with the R203W muation inserted into the Gt(ROSA)26Sor locus. The knock-in provides conditional, widespread expression of the mutant human PACS1 gene. Mice homozygous for the mutation are believed to be viable and fertile. Upon exposure to Cre recombinase, the LSL is removed and the human R203W mutation is expressed.

When combined with mice carrying cre expressed in neurons (B6.129S2-Emx1^tm1(cre)Krj/J, Stock No. 005628), double mutant progeny exhibit reduced dendritic spine density, increased dendritic arbor density, induced accumulation of varicosities, and reduced AMPAR-mediated glutamatergic excitatory mPSC amplitude and frequency. Western blot analysis and immunohistochemical staining confirmed that PACS1 is expressed at levels similar to endogenous PACS1 in the cortex and hippocampus.

Of note, the wild-type human PACS1 gene is distributed as C57BL/6J-Gt(ROSA)26Sor^{em2(CAG-PACS1)Gath}/J (Stock No. 038552). In addition, the orthologous mouse R201W mutation knocked in to the mouse Pacs1 locus is distributed as C57BL/6J-Pacs1^em2Gath/J (Stock No. 038550).

Development

CRISPR/cas9 endonuclease-mediated genome editing was used to insert hemagluttin-tagged human PACS1 cDNA with the R203W (arginine to tryptophan mutation), followed by an IRES and a GFP reporter under the control of the widespread CAG promoter into the Gt(ROSA)26Sor locus on chromosome 6. A loxP-flanked neomycin STOP precedes the PACS1 sequence. sgRNA, Cas9 endonuclease, and the donor targeting vector plasmid were introduced into C57BL/6J-derived zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6 mice for germline transmission. Founders were sequenced to confirm the mutation. The donating investigator indicates that the GFP is too low to be detected and was only used for PCR genotyping. The strain maintained on the C57BL/6 background. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{em1(CAG-PACS1*R203W)Gath}
Name: endonuclease-mediated mutation 1, Gary Thomas
MGI accession ID: MGI:7518813
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Humanized sequence; Epitope tag
Synonyms: PACS1^R203W; R26^{P1R203W; ROSA26-LSL-PACS1-R203W
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: C57BL/6J
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing is used to insert hemagluttin-tagged human PACS1 cDNA with the R203W (arginine to tryptophan mutation), followed by an IRES and a GFP reporter under the control of the widespread CAG promoter. A loxP-flanked neomycin STOP precedes the PACS1 sequence. The GFP is too low to be detected and was only used for PCR genotyping.}