Exons 5 and 6 of the mouse Nalcn gene are flanked by loxP sites. Cre-mediated excision of the floxed region results in a knock-out allele.
Huda Zoghbi, Baylor College of Medicine
Dejian Ren, University of Pennsylvania
In humans, mutations in the NALCN (sodium leak channel, non-selective) gene are associated with severe respiratory dysfunction. Deficiency of this gene causes lethal apnea in both humans and mice.
Exons 5 and 6 of the mouse Nalcn gene are flanked by loxP sites in this conditional mutant strain. Cre-mediated excision of the floxed region introduces a stop codon, resulting in a knock-out allele. The knock-out allele produces no detectable transcript, as determined by in situ hybridization and RT-PCR.
Crosses of these floxed mice with animals expressing cre recombinase from a range of respiratory network promoters revealed that Nalcn expression in glutamatergic neurons sustains neonatal respiration. Excision of the floxed region using a Vglut2-cre strain (see Stock No. 028863; also called Slc17a6tm2(cre)Lowl) results in 100% lethality.
KOMP embryonic stem (ES) cells (clone EPD0383_5_C01) were acquired from the KOMP Repository (Project ID: CSD70769). A cassette containing the following was introduced to intron 4: FRT-En2 SA-IRES-lacZ-pA-loxP-β actin-neomycin-pA-FRT-loxP. An additional loxP site was placed in intron 6. The mutation was created in C57BL/6N-Atm1Brd-derived JM8A3.N1 embryonic stem (ES) cells. Crosses with a strain expressing Flp recombinase were carried out to excise the lacZ/neo cassette, leaving exons 5 and 6 flanked by loxP sites. This line has been backcrossed to C57BL/6J for at least 10 generations by the donating laboratory.
|Allele Name||targeted mutation 1c, Wellcome Trust Sanger Institute|
|Allele Type||Targeted (Conditional ready (e.g. floxed))|
|Allele Synonym(s)||Nalcnflox; NALCNfx|
|Gene Symbol and Name||Nalcn, sodium leak channel, non-selective|
|Strain of Origin||C57BL/6N-Atm1Brd|
|Molecular Note||The L1L2_Bact_P cassette was inserted at position 123593515 of Chromosome 14 upstream of the critical exon(s) (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exon(s) at position 123592481. The "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele to remove the lacZ sequence and neo selection cassette, leaving the critical exons flanked by loxP sites. Subsequent cre expression results in a knockout mouse. Further information on targeting strategies used for this and other IKMC alleles can be found at http://www.informatics.jax.org/mgihome/nomen/IKMC_schematics.shtml.|
Homozygous and heterozygous floxed mice are viable and fertile.
When using the Nalcnflox mouse strain in a publication, please cite the originating article(s) and include JAX stock #030718 in your Materials and Methods section.
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