B6(Cg)-Ncf1^m1J/J

Stock number: 004742
Research areas: Hematological Research, Immunology, Inflammation and Autoimmunity Research, Mouse/Human Gene Homologs, Research Tools

Description

This strain is currently unavailable due to replenishing of cryopreserved stocks. Interested customers who register interest will be contacted when the strain is again available

 

Peritoneal neutrophils and macrophages, bone marrow cells and neutrophils isolated from bone marrow of mice homozygous for Ncf1^m1J spontaneous mutation fail to produce superoxide upon stimulation in vitro with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA).

Detailed description

Peritoneal neutrophils and macrophages, bone marrow cells and neutrophils isolated from bone marrow of mice homozygous for Ncf1^m1J fail to produce superoxide upon stimulation in vitro with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA), as determined by kinetic spectrophotometric analysis of cytochrome c reduction. Western blot analysis detected no full-length NCF1/p47^phox protein in cells from these mice; a faint band of slightly smaller molecular size than the wild type NCF1 protein was observed on probing with antibodies to NCF1. To exclude the possibility that the NCF1 protein is produced in cells of mutant mice but is degraded rapidly by endogenous proteases, bone marrow cells were isolated and samples prepared for western blot analysis in the presence of diisopropyl fluorophosphate (DFP); no difference was observed upon analysis of freshly prepared cell lysates made with and without DFP, indicating that NCF1 protein is not expressed in mutant cells (Huang et al. 2000).

Analysis for other NADPH oxidases involved in neutrophil superoxide production revealed that NCF2/p67^phox was present at wild type levels and CYBB/gp91^phox and CYBA/p22^phox were expressed at higher than wild type levels. Other neutrophil products were assayed and found not to differ in bone marrow cells of C57BL/6J vs. mutant mice: GR-1, a granulocyte marker present on approximately 40% of marrow cells by fluorescence activated cell sorting (FACS) analysis; NCF4/p40^phox and RAC1/RAC2 proteins, analyzed by western blot analysis; and expression of mRNAs encoding the granule proteins myeloperoxidase, elastase, cathepsin, lysozyme, lactoferrin and gelatinase as detected by reverse transcriptase - polymerase chain reaction (RT-PCR) (Huang et al. 2000).

The response of Ncf1^m1J/Ncf1^m1J mice to infectious or irritant (inflammatory stimulus) challenge has not been investigated (Huang et al. 2000). However, mice homozygous for the targeted mutation Ncf1^tm1Shl (Stock No. 027331) - which disrupts the gene near the point of the Ncf1^m1J mutation and, like the latter, results in failure of phagocytes to generate a superoxide burst following PMA stimulation in vitro - developed spontaneous infections with a variety of microorganisms including Staphylococcus xylosus, Lactobacillus, a hyaline septate mold and the fungus Paecilomyces, despite being maintained under specific pathogen free conditions. Ten-fold more Staphyloccoccus aureus bacteria survived intracellularly following in vitro uptake by phagocytes inblood from these mice than from control littermates, demonstrating a defect in the intracellular bactericidal activity of the mutant mice. Following intraperitoneal injection of the sterile irritant thioglycolate, twice as many leukocytes were recruited to the peritoneal cavities of Ncf1^tm1Shl homozygous mice than of littermate controls; proportions of neutrophils in mutant and control exudates were similar (Jackson et al., J Exp Med 182:751-758 1995).

Development

This spontaneous mutation arose in the B6.BKS(D)-Lepr^db/J (Stock No. 000697) production colony at The Jackson Laboratory. The mutation was identified in 1999. (Huang CK. et al. 2000) The mutations Lepr^db and m have been selectively bred out of this strain by crossing to C57BL/6J at least three times.

Allele

Symbol: Ncf1^m1J
Name: mutation 1, Jackson
MGI accession ID: MGI:2661962
Generation method: Spontaneous
Marker symbol: Ncf1
Marker name: neutrophil cytosolic factor 1
Chromosome: 5
Strain of origin: B6.Cg-Dock7^m +/+ Lepr^db/J
Molecular note: The mutation is an A-to-C transversion at the -2 position 5' off exon 8 in intron 7, changing splice acceptor site CAG to CCG. This results in aberrant splicing of the transcript. Immunoblotting detected no intact NCF1 protein in cells from these mice.