STOCK Prtn3^tm1.2Hrlu/J

Stock number: 030761
Product name: Prtn3^flox (or PR3^flox)
Research areas: Apoptosis Research, Cancer Research, Cell Biology Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

The Prtn3 floxed allele has loxP sites flanking exon 2 of the proteinase 3 gene. Removal of the floxed sequence creates a null allele. These mice may be useful for generating tissue-specific PR3-deletion for studying the role of this serine protease in modulating physiopathological inflammatory responses, including Caspase-3–mediated spontaneous/programmed cell death in neutrophils.

Detailed description

Programmed cell death (PCD) is a broad term encompassing apoptosis, autophagy, lysosomal-mediated PCD (LM-PCD) and necroptosis. Caspase-3-mediated spontaneous death in neutrophils is a prototype of PCD, and is critical for modulating physiopathological inflammatory responses. Cleavage and activation of caspase-3 in aging neutrophils is mediated by the serine protease proteinase 3 (PR3; encoded by Prtn3). Disruption of PR3 delays neutrophil death both in vitro and in vivo.

The Prtn3 floxed allele (Prtn3^flox) has loxP sites flanking Prtn3 exon 2. Mice homozygous for this floxed allele (Prtn3^flox/flox) are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator reports that homozygotes have normal expression levels of PRTN3 mRNA and protein.
When bred to mice that express Cre recombinase, the resulting offspring will have the floxed region deleted in cre-expressing tissues; creating the knock-out allele (Prtn3^-).
For example, when Prtn3^flox are bred to germline Cre-expressing mice, the resulting PR3 global knockout cells consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in an E. coli infection model of murine peritonitis [Loison et al. 2014 J Clin Invest 124:4445].

Development

The Prtn3 floxed allele was created for Dr. Hongbo R. Luo (Boston Children's Hospital / Harvard Medical School) by Ozgene (Australia) to have a loxP site upstream of exon 2, and a frt-flanked reverse-orientated PGK-neo-pA cassette followed by a second loxP site just downstream of exon 2 of the proteinase 3 locus (Prtn3) on chromosome 10. The construct was electroporated into B6.Cg-Thy1^a-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with albino C57BL/6 females (C57BL/6-Tyr^c-Brd/NCr) to establish the colony.

Mice were bred with FLPe-transgenic mice (on a C57BL/6 background) for germline deletion of the PGK-neo-pA cassette.

The donating investigator reported the resulting Prtn3 floxed mice were bred with C57BL/6 mice for several generations (see SNP note below) (and the FLPe-expressing transgene was removed) prior to sending males with black coat color to The Jackson Laboratory Repository in 2017. Upon arrival, sperm was cryopreserved. To establish our live colony in 2018, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
Of note, it is not known if these mice retained the coat color allele Tyr^c-Brd.

In 2018, 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 first generation rederived living colony at The Jackson Laboratory Repository. This revealed 3/43 markers throughout the genome were segregating for allele-type other than C57BL/6. In addition, all 5 C57BL/6 substrain markers were found to be heterozygous for C57BL/6NCrl;C57BL/6J allele-type. Collectively, these data suggest the mice had been backcrossed to C57BL/6NCrl for several generations and were ~88% C57BL/6N prior to sending to The Jackson Laboratory Repository. Our live colony in 2018 is ~93% C57BL/6 (mixed C57BL/6J and C57BL/6NCrl).

Allele

Symbol: Prtn3^tm1.2Hrlu
Name: targeted mutation 1.2, Hongbo R Luo
MGI accession ID: MGI:7446566
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Prtn3
Marker name: proteinase 3
Marker synonyms: ACPA; AGP7; CANCA; C-ANCA; MBN; MBT; mPR3; myeloblastin; NP4; NP-4; P29; PR3; PR-3
Chromosome: 10
Strain of origin: B6.Cg-Thy1^a
Molecular note: A loxP site was inserted upstream of exon 2 and a an FRT-flanked pgk-neo cassette and loxP site were inserted downstream of exon 2 via homologous recombination. Flp-mediated recombination removed the neo cassette.