STOCK Egln2^tm2Fong Egln1^tm2Fong Egln3^tm2Fong/J

Stock number: 028097
Product name: Phd1, Phd2, Phd3 Triple floxed
Research areas: Research Tools

Description

This Phd1^f/Phd2^f/Phd3^f triple floxed strain may be useful for generating conditional mutations in applications related to the study hypoxia inducible factor and oxygen homeostasis.

Detailed description

Prolyl hydroxylase domain (PHD) proteins catalyze oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor 1α and 2α subunits. Under normoxic conditions, hypoxia-inducible factor α subunits are targeted for degration by prolyl hydroxylation. The prolyl hydroxylase domain proteins encoded by Egln2, Egln1, and Egln3 have overlapping function in hydroxylating both HIF1A and HIF2A. These Phd1^f/Phd2^f/Phd3^f mice carry floxed alleles for the 3 genes. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have the floxed sequences of the 3 genes deleted in the cre-expressing tissues. Mice that are homozygous for the floxed mutations are viable and fertile. For each of these mutations, removal of the floxed sequence creates a null allele.

Development

This triple floxed mutant line was generated by breeding three independently created mutant lines, described below.



For the Egln2^tm2Fong (Egln2^floxN or Phd1^f) allele, a targeting vector designed by Dr. Guo-Hua Fong (Univ CT Health Center) containing a FRT site flanked NEO cassette was utilized. A loxP site and this selection cassette was inserted downstream of exon 3 of the targeted gene, and another loxP site was inserted upstream of exon 3. This construct was electroporated into (129S6/SvEvTac x C57BL/6NCrl)F1 derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were used to generate chimeras by tetraploid aggregation technique. Germline mice were crossed with transgenic mice expressing Flpe under the control of beta-actin promoter (on an unknown genetic background) to remove the FRT site flanked NEO cassette. The mice were then crossed to CD-1 mice to breed out the FLPe recombinase transgene.



For the Egln1^tm2Fong (Phd2^f) allele, a targeting vector designed by Dr. Guo-Hua Fong (Univ CT Health Center) containing a FRT site flanked NEO cassette was utilized. A loxP site and this selection cassette was inserted downstream of exon 2 of the targeted gene, and another loxP site was inserted upstream of exon 2. This construct was electroporated into (129S6/SvEvTac x C57BL/6NCrl)F1 derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were used to generate chimeras by tetraploid aggregation technique. The mice were then crossed to transgenic mice expressing Flpe recombinase under the control of beta-actin promoter (on an unknown genetic background) to remove the FRT site flanked NEO cassette. The mice were then crossed to CD-1 mice to breed out the FLPe recombinase transgene.



For the Egln3^tm2Fong (Phd3^f) allele, a targeting vector designed by Dr. Guo-Hua Fong (Univ CT Health Center) containing a FRT site flanked NEO cassette was utilized. A loxP site and this selection cassette was inserted downstream of exon 2 of the targeted gene, and another loxP site was inserted upstream of exon 2. This construct was electroporated into (129S6/SvEvTac x C57BL/6NCrl)F1 derived G4 embryonic stem (ES) cells which were transiently transfected with a FLPe recombinase vector to remove the NEO selection cassette. ES cells that had successfully undergone FLPe-mediated recombination and no longer retained the cassette were used to generate chimeras by tetraploid aggregation technique. The mice were then crossed to CD-1 mice.



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).

Allele

Symbol: Egln3^tm2Fong
Name: targeted mutation 2, Guo-Hua Fong
MGI accession ID: MGI:5431993
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Egln3
Marker name: egl-9 family hypoxia-inducible factor 3
Chromosome: 12
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: A loxP site was inserted upstream of exon 2. An additional loxP site and a neo cassette were inserted downstream of exon 2. This allele was used to generate Egln3^tm1Fong.

Allele

Symbol: Egln1^tm2Fong
Name: targeted mutation 2, Guo-Hua Fong
MGI accession ID: MGI:3778917
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Egln1
Marker name: egl-9 family hypoxia-inducible factor 1
Chromosome: 8
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: An frt-flanked neo cassette with a 5' loxP site was inserted downstream of exon 2 and an additional loxP site was inserted upstream of exon 2. Subsequent germ-line, cre-mediated recombination resulted in the creation of Egln1^tm1Fong.

Allele

Symbol: Egln2^tm2Fong
Name: targeted mutation 2, Guo-Hua Fong
MGI accession ID: MGI:5431991
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Egln2
Marker name: egl-9 family hypoxia-inducible factor 2
Chromosome: 7
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: A loxP site was inserted upstream of exon 3. An additional loxP and a neo cassette were inserted downstream of exon 3. This allele was used to generate Egln2^tm2Fong.