Pad4fl/fl floxed mice may be useful for studying the formation of neutrophil extracellular traps during acute infection.
Kerri Mowen, The Scripps Research Institute
Pad4fl/fl floxed mice possess loxP sites flanking exons 9-10 of the peptidyl arginine deiminase, type IV (Padi4) gene. These exons contain part of the PAD4 active site, as well as four additional residues that are essential for Ca2+ binding. PAD4 is an enzyme responsible for the citrullination and deimination conversion of arginine to citrulline residues on histones. PAD4-mediated deimination of histone H3 and H4 is required for the formation of neutrophil extracellular traps (NETs), specialized anti-microbial structures comprised of decondensed chromatin decorated with microbicidal agents. NETs are responsible for killing gram-positive and gram-negative bacteria as well as fungi. Increased amounts of NETs are detrimental in chronic neutrophil-mediated inflammation such as the lung diseases chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) due to increased severity of lung obstruction. Homozygous mice are viable and fertile. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have exons 9-10 deleted in the cre-expressing tissues.
When bred to B6.C-Tg(CMV-cre)1Cgn/J mice (Stock No. 006054) with widespread Cre recombinase expression, histone deimination is absent in neutrophils of resulting offspring, and they are defective in NET formation. PAD4 KO mice lose significantly more weight during influenza A infection, but retain normal survival. The number of lung infiltrating leukocytes was similar to that seen in wildtype mice during infection.
A targeting vector was designed by Dr. Kerri Mowen (The Scripps Research Institute) to insert a loxP site followed by a frt-flanked neomycin resistance (neo) cassette upstream of exon 9, and a second loxP site downstream of exon 10 of the peptidyl arginine deiminase, type IV (Padi4) gene. The construct was electroporated into B6.Cg-Thy1a-derived Bruce 4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into B6(Cg)-Tyrc-2J/J albino blastocysts and resulting chimeric males were bred with B6(Cg)-Tyrc-2J/J albino females (Stock No. 000058). Offspring were bred with B6.Cg-Tg(ACTFLPe)9205Dym/J transgenic mice (Stock No. 005703) to delete the neo cassette, and progeny were crossed to remove the Flp-expressing transgene. Pad4fl/fl mice were bred to C57BL/6J mice for at least 3 generations. 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). The Tyrc-2J allele was not maintained in the colony.
|Allele Name||targeted mutation 1.2, Kerri A Mowen|
|Allele Type||Targeted (Conditional ready (e.g. floxed))|
|Gene Symbol and Name||Padi4, peptidyl arginine deiminase, type IV|
|Strain of Origin||B6.Cg-Thy1a|
|Molecular Note||A targeting vector was designed to insert a loxP site followed by an FRT-flanked neomycin resistance (neo) cassette upstream of exon 9, and a second loxP site downstream of exon 10. Flp-mediated recombination removed the FRT-flanked neo cassette leaving exons 9 through 10 floxed.|
When maintaining a live colony, homozygous mice may be bred together.
When using the Pad4 flox mouse strain in a publication, please cite the originating article(s) and include JAX stock #026708 in your Materials and Methods section.