B6N.129(Cg)-Sema5a^tm1.2Alk/J

Stock number: 017533
Research areas: Developmental Biology Research, Sensorineural Research

Description

This knockout strain of the Sema5a gene exhibits no obvious abnormal phenotype, but when mated with a Sema5b knockout mouse, development of the neural tissue of the eye is impacted in the offspring.

Detailed description

In this knockout strain, exon 6 of the semaphorin 5A (Sema5a) gene is excised (includes coding region), abolishing gene function. Sema5A is a transmembrane protein which acts as a repellant for axonal guidance during retinal neural development. Wildtype P0 and P3 mice express Sema5A in the outer neuroblastic layer, directly adjacent to the inner neuroblastic layer (INBL). At P7, P10, and P14, the expression is only observed in the middle to outer part of inner nuclear layer (INL), and transcripts are not detectable at P21, when retinal development is almost complete. Homozygotes are viable and fertile. When bred to mice lacking Sema5b (Stock No. 017534), SEMA5A/B double KO mice exhibit severe defects in retinal ganglion cells, amacrine cells, cholinergic amacrine cells, calretinin-positive amacrine cells, and calbindin-positive amacrine cells. The defects lead to neurite mistargeting within both the inner plexiform layer (IPL) and INL resulting in functional defects in retinal responses to visual stimuli. These mice may be useful for studying axonal guidance and lamination regulation during retinal development.

Development

A targeting vector was designed to insert a loxP site upstream of exon 6, and a frt-flanked neomycin resistance (neo) cassette followed by a second loxP site just downstream of exon 6 of the semaphorin 5A (Sema5a) targeted gene. The construct was electroporated into 129-derived embryonic stem (ES) cells and correctly targeted ES cells were injected into C57BL/6NCrl blastocysts. Chimeric males were bred to C57BL/6NCrl females and the resulting offspring were subsequently bred to mice expressing CMV-cre to remove exon 6 and the neo cassette. These mice were then backcrossed to C57BL/6NCrl mice for at least 7 generations (see SNP note below). Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6NJ inbred mice (Stock No. 005304) for at least one generation to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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 rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Sema5a^tm1.2Alk
Name: targeted mutation 1.2, Alex L Kolodkin
MGI accession ID: MGI:5284881
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Sema5a
Marker name: sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A
Chromosome: 15
Strain of origin: 129
Molecular note: A loxP site was inserted upstream of exon 6 and an FRT flanked inverted neo cassette and second loxP site were inserted downstream of exon 6 via homologous recombination. Recombinase mediated recombination removed the neo cassette and exon 6. Western blot analysis confirmed the absence of protein expression in homozygous mice.