C57BL/6-Nes^{em1(flpo)Awar}/J

Stock number: 040639
Product name: PRISM
Strain synonyms: Nes-LSL-Flpo
Research areas: Research Tools

Description

PRISM knock-in/knock-out mice are designed to have a rox- and loxP-flanked destabilized green fluorescent protein (d4GFP) sequence and a loxP-flanked STOP cassette inserted at the start codon of the nestin (Nes) gene upstream of a flpo recombinase sequence. The insertion abolishes endogenous Nes expression. Cre- and Dre-mediated recombination results in the excision of the STOP sequence, enabling the tissue dependent expression of Flp in neural progenitors. These mice are useful for fate mapping genetically defined progenitors when used in conjunction with a Flp reporter.

Detailed description

PRISM knock-in/knock-out mice are designed to have a rox- and loxP-flanked destabilized green fluorescent protein (d4GFP) sequence and a loxP-flanked STOP cassette inserted at the start codon of the nestin (Nes) gene upstream of a flpo recombinase sequence. The insertion abolishes endogenous Nes expression. Nes encodes an intermediate filament protein expressed in the neural stem cells in the central and peripheral nervous system. Cre- and Dre-mediated recombination results in the excision of the STOP sequence, enabling the tissue dependent expression of Flp in neural progenitors.

Heterozygous mice are viable and fertile. It is unknown if homozygous mice are fertile.

The donating investigator bred these PRISM mice to EIIa-Cre mice (Stock No. 003724) to remove the STOP cassette in all cells, resulting in Flp recombinase being only dependent on Nestin expression. These mice were further crossed with a Flp reporter line, RC-FSF-LacZ, which expresses nuclear β-galactosidase upon removal of the frt-flanked stop cassette (FSF) by Flp recombinase. In the EIIa-Cre+, Nes-LSL-Flpo+, RC-FSF-LacZ+ brains, nβGal+ is observed in cells throughout the entire brain. No apparent leakiness is observed in the Nes-LSL-Flpo mice in the absence of Cre, and no nβGal staining was observed in littermate controls lacking Cre.

EGFP fluorescence is undetectable. The d4GFP cassette, being flanked by rox sites, can easily be removed using a Dre deleter mouse.

Development

Two sgRNAs were used to target the start codon of the nestin (Nes) gene for insertion of (from 5' to 3') a loxP site, a rox site, a destabilized green fluorescent protein (d4GFP) sequence, a SV40 polyadenylation signal, a second rox site, a stop cassette, a second loxP site, flpo, and a bGH polyadenylation signal. d4GFP is thus flanked by rox sites and can be removed by crossing to a Dre-deleter mouse. Cre excises the d4GFP and stop cassette, allowing Flpo expression when Nestin is expressed. sgRNA, single-stranded DNA (ssDNA), and Cas9 protein were introduced into the cytoplasm of fertilized eggs with well recognized pronuclei from C57BL/6 mice. Targeted embryos were transferred to pseudopregnant females, and correctly targeted Nes-LSL-Flpo or PRISM pups were identified by PCR and sequencing. The donating investigator reports the mice were backcrossed to C57BL/6 mice for an unknown number of generations. It is unknown if any off-target CRISPR sites are present. Cryopreserved sperm was sent to The Jackson Laboratory. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

Allele

Symbol: Nes^{em1(flpo)Awar}
Name: endonuclease-mediated mutation 1, Rajeshwar Awatramani
MGI accession ID: MGI:6740241
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed); Recombinase-expressing; Reporter
Synonyms: Nes-LSL-Flpo; PRISM
Marker symbol: Nes
Marker name: nestin
Marker synonyms: ESTM46; Ifaprc2; Marc2; Nbla00170; RC2
Chromosome: 3
Strain of origin: C57BL/6
Molecular note: Using the CRISPR/Cas9 technology, the Nestin locus in ES cells were targeted to insert loxP, rox, d4GFP, SV40 poly-A, rox, stop cassette, loxP, Flpo, and bGH poly-A at the start codon of the mouse Nes gene. d4GFP is flanked by rox sites and can be removed by crossing to a Dre-deleter mouse. Cre excises the d4GFP and stop cassette, allowing Flpo expression when Nestin is expressed.