B6.129(Cg)-Ntn1^tm1.2Tek/J

Stock number: 028070
Product name: netrin-1 KO
Research areas: Cardiovascular Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

This Ntn1 (netrin-1) knockout strain exhibits embryonic lethality and impaired axonal migration in the spinal cord. These mice may be suitable for use in studies related to cell migration, axon guidance, and angiogenesis.

Detailed description

The Ntn1 gene encodes a laminin-related secreted protein that can act as chemoattractant or chemorepellent and is important in axon guidance and cell migration during development. These mice carry a knock out allele of the Ntn1 gene, in which the first protein coding exon has been excised. No gene product (protein) is detected by Western blot analysis of spinal cord homogenates from homozygous E14.5 embryos. The floxed allele was not detected by RT-PCR of homozygous embryos. Homozygotes die around E14.5. Mice that are heterozygous for the targeted mutation are viable and fertile. Between E10.5 and E14.5, homozygous embryos exhibit absence of ventral spinal commissure, and disorganized dorsal root entry zone.

Development

A targeting vector containing a FRT site flanked Neo selection cassette was utilized in the construction of this mutant. This selection cassette and a loxP site was inserted downstream of the first protein coding exon of the targeted gene, and another loxP site was inserted upstream of the first protein coding exon. This construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺ derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting netrin-1^flox/+ chimeric animals were crossed to mice expressing FLP1 recombinase under the control of the human beta actin promoter (Stock No. 003800). Mice that no longer carried the NEO cassette and retained the loxP site flanked first protein coding exon were then bred to C57BL/6 mice for 7 generations to remove the Tg(ACTFLPe)9205Dym transgene. The mice were then bred to germline Cre deleter transgenic mice (Stock No. 006054) to excise the first protein coding exon. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

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, all 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 C57BL/6N genetic background.

Allele

Symbol: Ntn1^tm1.2Tek
Name: targeted mutation 1.2, Timothy E Kennedy
MGI accession ID: MGI:5752968
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Ntn1
Marker name: netrin 1
Chromosome: 11
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: The targeting vector contains a FRT-flanked Neo selection cassette and a loxP site was inserted downstream of the first protein coding exon of the targeted gene, and another loxP site was inserted upstream of the first protein coding exon. Flp-mediated recombination removed the FRT-flanked neo cassette. Cre-mediated recombination removed the first protein coding exon. Western blot and immunohistochemical analysis of spinal cord confirmed the absence of protein expression.