STOCK Nphs1^tm1.1Pgarg/J

Stock number: 032133
Product name: Nphs1^fl , Nphs1 flox , nphs1 floxΔneo
Research areas: Cell Biology Research, Developmental Biology Research, Diabetes and Obesity Research, Endocrine Deficiency Research, Internal/Organ Research, Research Tools

Description

129Sv.Nphs1^fl mice have loxP sites flanking exon 5 of the nephrosis 1, nephrin gene (Nphs1). Removal of the floxed sequence results in expression of a protein truncated at the second extracellular IgG domain without the transmembrane or intracellular domains. 129Sv.Nphs1^fl mice may be useful for studying kidney podocyte survival and the glomerulus, as well as glucose-stimulated insulin responses and islet survival.

Detailed description

Nephrin (Nphs1) is a member of a family of cell adhesion modulating proteins. Nephrin in the kidneys regulates glomerular vascular permeability. Nphs1 mutations in human or deletion in research animals results in a developmental failure of foot process formation. Nephrin is also expressed in pancreatic islets, where it plays a role in beta-cell function and survival.

The Nphs1^fl allele has loxP sites flanking Nphs1 exon 5 (encoding nucleotide residues 20805-21301 [ENSMUST00000006825]). When on a 129Sv/J genetic background, mice heterozygous or homozygous for this floxed allele (Nphs1^fl/+ or Nphs1^fl/fl, respectively) are viable and fertile with no reported gross physical or behavioral abnormalities.

Upon Cre recombinase exposure, the deletion of Nphs1 exon 5 causes a frameshift mutation immediately after exon 4 - resulting in premature stop codon that terminates translation after the first 196 amino acids (at the second extracellular IgG domain). The donating investigator predicts that both known Nphs1 isoforms will express a truncated protein that lacks the transmembrane and intracellular domains, and as such cannot be inserted at the membrane.
For example, when Nphs1^fl are bred to 129Sv-congenic NPHS2-Cre mice (same Tg(NPHS2-cre)295Lbh transgene as Stock No. 008523), the resulting Nphs1^fl/fl, podocin-creTg/+ animals have podocyte-specific ablation of NPHS1 function - leading to aberrant podocyte foot processes, massive proteinuria at ~1 week of age and death at ~2 weeks of age.
Furthermore, when Nphs1^fl are bred to NPHS2-iCreER(T2) mice (see Tg(NPHS2-icre/ERT2)1Dnsh), the resulting Nphs1^Tam-cre animals (Nphs1^{fl/fl,iCre(ER)T2}) have tamoxifen-inducible, podocyte-specific ablation of NPHS1 function - allowing the study of nephrin-deletion in mature glomeruli.

Development

The Nphs1^fl (floxed exon 5) allele was created by Dr. Puneet Garg (University of Michigan). First, a targeting vector was designed to insert a loxP site upstream of exon 5, and a frt-PGK-neo-frt-loxP cassette downstream of exon 5 of the nephrosis 1, nephrin locus (Nphs1) on chromosome 7. Nphs1 exon 5 encodes nucleotide residues 20805-21301 [ENSMUST00000006825]. The targeting vector was introduced into the pronuclei of fertilized oocytes from 129/SvJ mice. Chimeric animals were bred to FlpE-expressing 129/SvJ mice (no additional details) for germline deletion of the neo cassette. The FlpE-expressing transgene was later removed. The donating investigator reports the Nphs1 floxΔneo (Nphs1^fl ; floxed exon 5) colony was maintained on a 129Sv genetic background (see SNP note below), and males (agouti coat color) were sent to The Jackson Laboratory Repository in 2019. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize 129S1/SvImJ oocytes (Stock No. 002448).

Of note, it is not known if the Y chromosome has been fixed to the 129S background in this Nphs1^fl mouse line.



In 2020 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 rederived living colony at The Jackson Laboratory Repository. Many of the 43 markers throughout the genome were segregating with an unknown source.

Allele

Symbol: Nphs1^tm1.1Pgarg
Name: targeted mutation 1.1, Puneet Garg
MGI accession ID: MGI:6187990
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Synonyms: Nphs1^fl
Marker symbol: Nphs1
Marker name: nephrosis 1, nephrin
Marker synonyms: CNF; nephrin; NPHN
Chromosome: 7
Strain of origin: 129
Molecular note: A loxP site was inserted upstream of exon 5 and an FRT flanked neo cassette and loxP site were inserted downstream of exon 5 via homologous recombination. Flp mediated recombination removed the neo cassette.