STOCK Cfd^{tm1.1(CFD)Song}/J

Stock number: 040653
Product name: hFD^KI
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

hFD^KI/KI knock-in mice have exons 2 to 4, and portions of exons 1 and 5, of the endogenous Cfd gene replaced with the entire human CFD cDNA coding sequence. These mice are useful for studying alternative pathway complement activity as they develop complement-mediated C3 glomerulopathy (C3G) characterized by glomerular C3 deposition, proteinuria, crescentic glomerulonephritis, and renal failure. They can be used to identify new roles of FD in vivo and provide a new platform for screening of human FD-targeted drugs.

Detailed description

hFD^KI/KI knock-in mice have exons 2 to 4, and portions of exons 1 and 5, of the endogenous complement factor D (Cfd) gene replaced with the entire human CFD cDNA coding sequence. Also known as FD, Cfd encodes a serine peptidase that catalyzes the cleavage of factor B which then becomes the C3 convertase, the rate-limiting step of the alternative pathway of the complement system of innate immunity. The complement pathway plays an essential role in the migration of cells during development and in innate and adaptive immune responses that help the body clear foreign material by recognizing pathogens and neutralizing them.

In cells and in circulation, CFD enables the creation of C3 which leads to formation of the membrane attack complex and lysis of the target cell, both of which contribute to tissue injury and pathogen recognition and elimination. The complement system plays an important role in the pathogenesis of C3 glomerulopathy (C3G), a rare kidney disease caused by dysregulated alternative pathway complement activation characterized by glomerular C3 deposition, proteinuria, crescentic glomerulonephritis, and renal failure. Mutations in CFD can lead to complement factor D deficiency, increasing susceptibility to bacterial meningitis.

hFD^KI/KI homozygous knock-in mice produce human FD protein in adipose tissue and blood but not mouse FD protein. hFD exhibits alternative pathway complement activity in 50% of plasma and activity is also detected in the urine. When crossed with complement component factor h (Cfh) knock-out mice (fH^m/m) and properdin^-/- knock-out mice (Stock No. 040652), resulting FH^m/mP^-/-hFD^KI/KI mice have an earlier onset of C3G disease that progressed more rapidly than observed in FH^m/mP^-/- mice.

These hFD^KI/KI knock-in can be used to identify new roles of FD in vivo and provide a new platform for screening of human FD-targeted drugs.

Homozygous mice are viable and fertile.

Development

The hFD^KI targeting vector was designed to replace exons 2 to 4, and portions of exons 1 and 5, of the endogenous complement factor D (Cfd) gene with entire human CFD cDNA coding sequence (826 bp). An FRT-flanked neomycin resistance (neo) cassette was inserted downstream of the human CFD sequence. A nonsense C-to-G mutation was introduced in the human cDNA to destroy an internal XhoI site and an additional XhoI site was introduced into the 3' UTR of the endogenous exon 5. The construct was electroporated into B6.Cg-Thy1^a-derived Bruce 4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into BALB/c blastocysts and the resulting chimeric males were bred to C57BL/6J females (Stock No. 000664). Resulting hFD^KI mice were bred to ACTB:FLPe mice (Stock No. 005703) to remove the neo cassette. Mice lacking the neo cassette were maintained on the C57BL/6J genetic background by the donating investigator (see SNP note below). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.



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. Six of the 43 markers throughout the genome were segregating with an unknown source. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed genetic background.

Allele

Symbol: Cfd^{tm1.1(CFD)Song}
Name: targeted mutation 1.1, Wen-Chao Song
MGI accession ID: MGI:8217213
Generation method: Targeted
Attributes: Humanized sequence
Synonyms: hFD^KI
Marker symbol: Cfd
Marker name: complement factor D (adipsin)
Marker synonyms: ADIPSIN; Adn; D component (adipsin) of complement; DF; EVE; factor D; PFD
Chromosome: 10
Strain of origin: B6.Cg-Thy1^a
Molecular note: The targeting vector was designed to replace exons 2 to 4, and portions of exons 1 and 5, of the endogenous complement factor D (Cfd) gene with the entire human CFD cDNA coding sequence (826 bp). An FRT-flanked neomycin resistance (neo) cassette was inserted downstream of the human CFD sequence. A nonsense C-to-G mutation was introduced in the human cDNA to destroy an internal XhoI site, and an additional XhoI site was introduced into the 3' UTR of the endogenous exon 5. FLPe recombinase removed the neo cassette.