B6;129S6-Ctxn3^{em1(flpo)Tasic}/J

Stock number: 034303
Product name: Ctxn3-IRES2-FlpO-neo
Research areas: Neurobiology Research, Research Tools

Description

Ctxn3-IRES2-FlpO-neo knock-in mice are designed to have optimized FLP recombinase expression directed to cortexin 3-expressing cells. FlpO expression/activity is observed in many locations throughout brain, with especially strong expression in olfactory bulb, amygdala, medial habenula, thalamus, cerebellum, and laminar expression patterns in cortex that vary across cortical areas. These mice may be used to generate conditional mutations for studying the role of CTXN3 in brain disorders (e.g., schizophrenia, GABAergic neurotransmission).

Detailed description

The Ctxn3-IRES2-FlpO-neo knock-in mutation has an IRES2 sequence and an optimized FLP recombinase (FlpO) gene inserted immediately downstream of the Ctxn3 STOP codon - this is designed to have the endogenous cortexin 3 promoter/enhancer elements direct FlpO expression to CTXN3+ cells. When Ctxn3-IRES2-FlpO-neo mice are bred with mice containing frt-flanked sequences, FlpO-mediated recombination will result in deletion of the frt-flanked sequences in Ctxn3-expressing cells in the offspring.

The donating investigator reports FlpO recombinase expression/activity largely recapitulates the endogenous Ctxn3 gene. Specifically, they report expression in many locations throughout brain, with especially strong expression in olfactory bulb, amygdala, medial habenula, thalamus, cerebellum, and laminar expression patterns in cortex that vary across cortical areas. To date (April 2021), they have observed FlpO expressing cells mainly in layer 6 intratelencephalic (IT) and corticothalamic (CT) excitatory neurons in primary visual cortex (they have not yet profiled neurons for other brain regions to pinpoint cellular identity of the labeled cells). The donating investigator did not examine FlpO expression in tissues other than brain.

This knock-in allele is designed to avoid disrupting expression of the endogenous protein, although Ctxn3 expression from the Ctxn3-IRES2-FlpO-neo allele has not been evaluated to date (April 2021).

Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. It has not been attempted to make this strain homozygous to date (April 2021).

Development

The Ctxn3-IRES2-FlpO-neo mice were created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) via CRISPR/Cas9 methods to have an IRES2 sequence and an optimized FLP recombinase (FlpO) gene inserted immediately following the STOP codon of the cortexin 3 gene (Ctxn3) on chromosome 18. The development of this strain included Addgene Plasmid #181769 and #181770. The specific details are below.

The guide vector contained the Ctxn3 guide RNA sequences designed for the endogenous stop codon and also a DNA sequence encoding a human codon-optimized Streptococcus pyogenes CRISPR associated protein 9 (SpCas9). Of note, the guide vector is designed so the SpCas9 protein is only expressed in embryonic stem (ES) cells and the SpCas9 DNA sequence does not integrate into the genome.

The donor plasmid contained an internal ribosome entry site 2 sequence (IRES2; allows translation initiation in the middle of an mRNA sequence), a FlpO recombinase gene (a codon-optimized FLPe gene modified for translation in mammalian cells and higher recombination efficiency in cells and in mice), a bovine growth hormone polyA sequence, an attB site, a PGK/gb2 promoter-Neomycin resistance gene-PGK polyA cassette and an attP site - all flanked by Ctxn3 homology arms.

The guide vector and donor plasmid were co-electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 ES cells. Chimeric mice were generated by aggregating ES cells with morula-stage embryos, and the resulting chimeras were then bred to C57BL/6J mice to achieve germline transmission. The resulting Ctxn3-IRES2-FlpO-neo colony was bred with C57BL/6J for several generations. In 2020, males from generation N2 (black coat color) were sent to The Jackson Laboratory. Upon arrival, males are used to cryopreserve sperm. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Ctxn3^{em1(flpo)Tasic}
Name: endonuclease-mediated mutation 1, Bosiljka Tasic
MGI accession ID: MGI:6514362
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Synonyms: Ctxn3^em1(flpo)Hze
Marker symbol: Ctxn3
Marker name: cortexin 3
Marker synonyms: ENSMUSG00000069372; KABE
Chromosome: 18
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: CRISPR/cas9 genome editing is used to insert an internal ribosome entry site 2 sequence, a FlpO recombinase gene, a bovine growth hormone polyA sequence, an attB site, a PGK/gb2 promoter-Neomycin resistance gene-PGK polyA cassette and an attP site immediately following the STOP codon. The guide vector is designed so the SpCas9 protein is only expressed in embryonic stem (ES) cells and the SpCas9 DNA sequence does not integrate into the genome.