B6;129S6-Slc17a7^{em1(flpo)Tasic}/J

Stock number: 034422
Product name: Slc17a7-IRES2-FlpO-WPRE-neo [or Vglut1-IRES2-FlpO-WPRE-neo]
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Slc17a7-IRES2-FlpO-WPRE-neo (or Vglut1-IRES2-FlpO-WPRE-neo) knock-in mice are designed to have optimized FLP recombinase expression directed to VGLUT1-expressing cells. The donating investigator reports pan-excitatory FlpO expression/activity in brain, broadly in all neocortical excitatory neurons. These mice may be used to generate conditional mutations for studying glutamatergic synaptic vesicle trafficking and vesicle-bound, sodium-dependent phosphate transportation.

Detailed description

Vesicular glutamate transporter 1 (VGLUT1; encoded by Slc17a7) is a vesicle-bound, sodium-dependent phosphate transporter expressed in the neuron-rich regions of the brain, is preferentially associated with the membranes of synaptic vesicles and functions in glutamate transport.

The Slc17a7-IRES2-FlpO-WPRE-neo (or Vglut1-IRES2-FlpO-WPRE-neo) knock-in mutation has an IRES2 sequence and an optimized FLP recombinase (FlpO) gene inserted immediately downstream of the Slc17a7 STOP codon - this is designed to have the endogenous promoter/enhancer elements direct FlpO expression to VGLUT1+ cells. When Slc17a7-IRES2-FlpO-WPRE-neo mice are bred with mice containing frt-flanked sequences, FlpO-mediated recombination will result in deletion of the frt-flanked sequences in Slc17a7-expressing cells in the offspring.

The donating investigator reports FlpO recombinase expression/activity largely recapitulates the endogenous Slc17a7 gene. Specifically, they report pan-excitatory FlpO expression/activity in brain, broadly in all neocortical excitatory neurons. 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 Slc17a7 expression from the Slc17a7-IRES2-FlpO-WPRE-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 Slc17a7-IRES2-FlpO-WPRE-neo (or Vglut1-IRES2-FlpO-WPRE-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 solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter) gene (Slc17a7; Vglut1) on chromosome 7. The development of this strain included Addgene Plasmid #181775 and #181776. The specific details are below.

The guide vector contained the Slc17a7 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 woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), 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 Slc17a7 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 donating investigator reports the resulting Slc17a7-IRES2-FlpO-WPRE-neo colony was bred with C57BL/6J for several generations. In 2020, males from generation N4 (black and agouti coat colors) were sent to The Jackson Laboratory. Upon arrival, this colony was bred to C57BL/6J (Stock No. 000664) for at least one generation to remove the rd8 mutation (see SNP note below), after which sperm was cryopreserved.
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6J background during backcrossing.

In 2020, 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 males sent to The Jackson Laboratory Repository. Most of the markers were C57BL/6J allele-type and some were C57BL/6;129 (as expected). Importantly, at least one of the markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the agouti male was segregating for the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6;129 genetic background (as expected) with a possible C57BL/6N contribution. The rd8 mutation was removed from the colony, after which sperm was cryopreserved.

Allele

Symbol: Slc17a7^{em1(flpo)Tasic}
Name: endonuclease-mediated mutation 1, Bosiljka Tasic
MGI accession ID: MGI:6514364
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Synonyms: Slc17a7^em1(flpo)Hze
Marker symbol: Slc17a7
Marker name: solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 7
Marker synonyms: 2900052E22Rik; BNPI; Vglut1
Chromosome: 7
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 woodchuck hepatitis virus post-transcriptional regulatory element, a bovine growth hormone polyA sequence, an attB site, a PGK/gb2 promoter-Neomycin resistance gene-PGK polyA cassette and an attP site following the STOP codon. The guide vector contained the Slc17a7 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.