B6.Cg-Htra3^tm1.1Hohj Htra2^tm1.2Hohj Htra1^tm2.1Hohj Htra4^tm1.2Hohj/J

Stock number: 039034
Product name: Htra1 KO, Htra2 KO, Htra3 KO, Htra4 KO

Research areas: Cell Biology Research, Metabolism Research, Neurobiology Research, Research Tools

Description

This quadruple Htra knock-out has deletions in Htra1 exons 2-3, Htra2 exons 2-4, Htra3 exon 3, and Htra4 exons 4-6. This strain may be useful in studies of age-related diseases including Parkinson's disease and age-related macular degeneration.

Of note, The floxed version of Htra2 is available as C57BL/6J-Htra2^tm1.1Hohj/J, Stock No. 039032.

Detailed description

Htra1, Htra2, Htra3, and Htra4 are members of the high-temperature requirement A (HtrA) family of stress proteins. All HTRA genes have four protein domains. HTRA1,3, and 4 have an insulin-like growth factor binding domain, a kazal domain, a trypsin-like peptidase domain, and a PDZ domain. HTRA2 differs in a mitochondrial localization sequence, a transmembrane domain and inhibitor of apoptosis binding domain in the N-terminus but contains the peptidase and PDZ domains. HTRA1 is involved in protein quality control. GWAS studies have identified the region containing HTRA1 and ARMS2 (10q26) with increased susceptibility to age-related macular degeneration. HTRA2 is involved in mitochondrial stress signaling under physiological conditions and acts as a stimulator of apoptosis under extreme conditions. Loss of Htra2 results in striatal neuronal loss, a parkinsonian phenotype, and death by 40 days of age. Dysregulation of HTRA3 is associated with preeclampsia and some cancers. Up-regulation of HTRA4 is associated with preeclampsia.

This quadruple Htra knock-out has deletions in Htra1 exons 2-3, Htra2 exons 2-4, Htra3 exon 3, and Htra4 exons 4-6. All deletions result in null alleles. Mice homozygous for the Htra1, Htra3, and Htra4 knock-outs and heterozygous for Htra2 are viable and fertile. Homozygous Htra2 knock-out mice exhibit striatal neuronal loss, a parkinsonian phenotype, and death by 40 days of age. This strain may be useful in studies of age-related diseases including Parkinson's disease and age-related macular degeneration.

Of note, The floxed version of Htra2 is available as C57BL/6J-Htra2^tm1.1Hohj/J, Stock No. 039032.

Development

Mice carrying the knock-out alleles for Htra1, Htra2, Htra3, and Htra4 were combined to create the quadruple knock-out.

The Htra1 targeting vector was designed to insert a loxP site upstream of exon 2, and an FRT-flanked PGK-neomycin cassette followed by a loxP-site downstream of exon 3 of the HtrA serine peptidase 1 (Htra1) locus on chromosome 7. The construct was electroporated into C57BL/6J derived embryonic stem (ES) cells (Ozgene). Correctly targeted ES cells were injected into blastocysts. The resulting chimeras were crossed to C57BL/6J mice. Mice were then crossed to C57BL/6 mice expressing a OzCre (Ozgene) to remove the exons 2-3 and the neo cassette. Mice were then bred to C57BL/6J to remove the cre allele.

The Htra2 targeting vector was designed to insert a loxP site upstream of exon 2, and a FRT-flanked PGK-neomycin cassette followed by a loxP-site downstream of exon 4 of the HtrA serine peptidase 2 (Htra2) locus on chromosome 6. The construct was electroporated into C57BL/6J derived embryonic stem (ES) cells (Ozgene). Correctly targeted ES cells were injected into blastocysts. The resulting chimeras were crossed to C57BL/6J mice. Mice were then crossed to C57BL/6 mice expressing a flpe (Rosa26FlpE/Ozgene) to remove the neo cassette and an OzCre (Ozgene) to remove exons 4-6. Mice were then bred to C57BL/6J to remove the flp and cre alleles.

The Htra3 targeting vector was designed to insert a loxP site upstream of exon 3, and an FRT-flanked PGK-neomycin cassette followed by a loxP-site downstream of exon 3 of the HtrA serine peptidase 3 (Htra3) locus on chromosome 5. The construct was electroporated into C57BL/6J derived embryonic stem (ES) cells (Ozgene). Correctly targeted ES cells were injected into blastocysts. The resulting chimeras were crossed to C57BL/6J mice. Mice were then crossed to C57BL/6 mice expressing a OzCre (Ozgene) to remove the exon 3 and the neo cassette. Mice were then bred to C57BL/6J to remove the cre allele.

The Htra4 targeting vector was designed to insert a loxP site upstream of exon 4, and an FRT-flanked PGK-neomycin cassette followed by a loxP-site downstream of exon 6 of the HtrA serine peptidase 4 (Htra4) locus on chromosome 8. The construct was electroporated into C57BL/6J derived Bruce4 embryonic stem (ES) cells (Ozgene). Correctly targeted ES cells were injected into blastocysts. The resulting chimeras were crossed to C57BL/6J mice. Mice were then crossed to C57BL/6 mice expressing a flpe (Rosa26FlpE/Ozgene) to remove the neo cassette and an OzCre (Ozgene) to remove exons 4-6. Mice were then bred to C57BL/6J to remove the flp and cre alleles.

All strains were maintained on the C57BL/6J background. This strain was cryopreserved using embryos.

Allele

Symbol: Htra4^tm1.2Hohj
Name: targeted mutation 1.2, Josephine Hoh
MGI accession ID: MGI:6266863
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Htra4
Marker name: HtrA serine peptidase 4
Marker synonyms: B430206E18Rik; RGD1306242
Chromosome: 8
Strain of origin: B6.Cg-Thy1^a
Molecular note: A loxP site was inserted upstream of exon 4. A FRT-flanked neomycin resistance cassette with a loxp site was inserted downstream of exon 5 and removed by flp-mediated recombination. A third loxP site was inserted downstream of exon 6. Cre-mediated recombination removed exons 4 through 6.

Allele

Symbol: Htra3^tm1.1Hohj
Name: targeted mutation 1.1, Josephine Hoh
MGI accession ID: MGI:7579338
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Htra3
Marker name: HtrA serine peptidase 3
Marker synonyms: 2210021K23Rik; 9530081K03Rik; Prsp; RGD1308120; Tasp
Chromosome: 5
Strain of origin: C57BL/6
Molecular note: The targeting vector is designed to insert a loxP site upstream of exon 3, and an FRT-flanked PGK-neomycin cassette followed by a loxP-site downstream of exon 3. Cre-mediated recombination removed exon 3 and the neo cassette.

Allele

Symbol: Htra2^tm1.2Hohj
Name: targeted mutation 1.2, Josephine Hoh
MGI accession ID: MGI:5752863
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: HTRA2 KO
Marker symbol: Htra2
Marker name: HtrA serine peptidase 2
Marker synonyms: HtrA2; MGCA8; mnd2; OMI; PARK13; PRSS25
Chromosome: 6
Strain of origin: C57BL/6J
Molecular note: A loxP site was inserted upstream of exon 2. An FRT-flanked neomycin resistance cassette with a 3' loxP site was inserted downstream of exon 4. Flp-mediated recombination removed the selection cassette. Cre-mediated recombination removed exons 2 through 4. Western blotting confirmed loss of protein in brain, spleen, and thymus tissues from homozygous mutant mice.

Allele

Symbol: Htra1^tm2.1Hohj
Name: targeted mutation 2.1, Josephine Hoh
MGI accession ID: MGI:7579347
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Htra1
Marker name: HtrA serine peptidase 1
Marker synonyms: ARMD7; CADASIL2; CARASIL; CARASIL2; HtrA; HtrA1; insulin-like growth factor binding protein 5 protease; L56; ORF480; Prss11; RSPP11
Chromosome: 7
Strain of origin: C57BL/6
Molecular note: The targeting vector is designed to insert a loxP site upstream of exon 2, and an FRT-flanked PGK-neomycin cassette followed by a loxP-site downstream of exon 3. Cre-mediated recombination removed the neo cassette and exons 2-3.