B6.Cg-Flrt2^em1Jnk/J

Stock number: 038570
Product name: FLRT2^UF , FLRT2^H170N
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research, Sensorineural Research

Description

FLRT2^UF mice carry a point mutation that disrupts interactions between fibronectin leucine rich transmembrane protein 2 (FLRT2) and its ligands of the unc-5 netrin receptor family (UNC5A, UNC5B, UNC5C, and UNC5D). This strain has been useful in studies of selective synaptic partnering by neurons and FLRT2-UNC5 signaling.

Detailed description

During retinal development, fibronectin leucine rich transmembrane protein 2 (FLRT2) is expressed by neurons of the retinal direction-selective (DS) circuit, while its ligands unc-5 netrin receptors C (UNC5C) and D (UNC5D) are expressed by GABAergic neurons that are not part of this circuit. Direct FLRT2-UNC5 binding prevents intermingling of DS circuit dendrites with non-circuit partners in the retinal inner plexiform layer.

These FLRT2^UF mice carry a CRISPR/cas9-generated H170N point mutation that adds a N-glycosylation site at the UNC5-binding surface. The mutation disrupts interactions between FLRT2 and its UNC5-family ligands (UNC5A, UNC5B, UNC5C, and UNC5D) without altering trafficking, surface expression, or interactions with latrophilins. When FLRT2-UNC5C transcellular signals are disrupted, retinal ganglion cells of the DS circuit generate ectopic dendrites that are uncoupled from their circuit partners, the starburst amacrine cells, instead acquiring numerous inhibitory synapses from other amacrine cells. Disruption of FLRT2-UNC5D binding causes starburst amacrine cells to produce ectopic dendrites.

Homozygous mice on a C57BL/6J background (backcross generation N4-5 or more) typically die shortly after birth, though some survive. The fertility of surviving homozygotes has not been tested. The donating laboratory maintained this line through heterozygote x C57BL/6J (Stock No. 000664) crosses. Their "mixed background" experimental animals were created by crossing heterozygous mice to SJL/J (Stock No. 000686), thereby generating hybrid animals that were used to generate homozygous mutants for experiments. These mutants on the C57BL/6J-SJL/J mixed background were not used for colony maintenance, but are viable, fertile, and without overt phenotype.

Development

CRISPR/Cas9 methodologies were used to introduce an H170N (nucleotide change CAC to AAT) in exon 2 of the mouse Flrt2 gene in B6SJLF1 embryos. A single guide RNA (sgRNA) was produced in which the PAM sequence overlaps the H170 codon (sequence 5' GCCCAACAGGCACGCTACTCagg; PAM site is denoted by lowercase letters). The PAM sequence is destroyed by the codon-altering mutation, thereby rendering correctly edited alleles immune to further cas9 cutting activity. Overall, the strain was backcrossed to C57BL/6J for a total of 6 times by the donating laboratory. Mice were bred to C57BL/6J-progressed B6.Cg-Tg(Hlxb9-GFP)1Tmj/J mice (original Stock No. 005029) for one generation early on, then backcrossed to C57BL/6J for 4 generations before shipment to The Jackson Laboratory. The Tg(Hlxb9-GFP)1Tmj allele was bred out of the colony.

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve C57BL/6J (Stock No. 000664). The donating laboratory maintained this "C57BL/6 background" line through heterozygote x C57BL/6J (Stock No. 000664) crosses. Their "mixed background" experimental animals were created by crossing heterozygous mice to SJL/J (Stock No. 000686) hybrid animals. Mixed background animals were not used for colony maintenance.

In 2023, 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. One of the 43 markers, on Chromosome 12, was segregating. This was likely a passenger segment from Flrt2 mutation.

Allele

Symbol: Flrt2^em1Jnk
Name: endonuclease-mediated mutation 1, Jeremy Kay
MGI accession ID: MGI:7505615
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: FLRT2^H170N; FLRT2^UF
Marker symbol: Flrt2
Marker name: fibronectin leucine rich transmembrane protein 2
Chromosome: 12
Strain of origin: (C57BL/6 x SJL)F1
Molecular note: CRISPR/cas9 methodologies are used to introduce an H170N (nucleotide change CAC to AAT, note AAC was originally reported) in exon 2. The mutation disrupts interactions between FLRT2 and its ligands UNC5C and UNC5D. A single guide RNA (sgRNA) was produced in which the PAM sequence overlaps the H170 codon (sequence 5' GCCCAACAGGCACGCTACTCagg; PAM site is denoted by lowercase letters). The PAM sequence is destroyed by the codon-altering mutation, thereby rendering correctly edited alleles immune to further cas9 cutting activity.