Limits...
New Strategy of Functional Analysis of PHGPx Knockout Mice Model Using Transgenic Rescue Method and Cre-LoxP System.

Imai H - J Clin Biochem Nutr (2009)

Bottom Line: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids.PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription.All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane Minato-ku Tokyo 108-8641, Japan.

ABSTRACT
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.

No MeSH data available.


Related in: MedlinePlus

Our established transgenic rescued floxed PHGPx knockout mice (Control mice) and tissue specific conditional PHGPx knockout mice by deletion of PHGPx-loxP transgene using Cre-loxP system. We established three lines of PHGPx-loxP TG/KO mice (TG KO, Control mice), all of which were viable and fertile, with body weights indistinguishable from those of wild-type mice. The PHGPx-loxP transgene could transcribe only PHGPx mRNA in the PHGPx-loxP TG/KO mice at nearly the same level as in the wild-type mice. Using a Cre-loxP conditional knockout strategy, we could generate tissue-specific PHGPx knockout mice (Cre TG KO mice) by mating PHGPx-loxP TG/KO mice with tissue-specific Cre expressing PHGPx heterozygous mice. In tissue specific PHGPx knockout mice, PHGPx-loxP transgene was specifically disrupted by tissue specific Cre recombinase.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2803127&req=5

Figure 6: Our established transgenic rescued floxed PHGPx knockout mice (Control mice) and tissue specific conditional PHGPx knockout mice by deletion of PHGPx-loxP transgene using Cre-loxP system. We established three lines of PHGPx-loxP TG/KO mice (TG KO, Control mice), all of which were viable and fertile, with body weights indistinguishable from those of wild-type mice. The PHGPx-loxP transgene could transcribe only PHGPx mRNA in the PHGPx-loxP TG/KO mice at nearly the same level as in the wild-type mice. Using a Cre-loxP conditional knockout strategy, we could generate tissue-specific PHGPx knockout mice (Cre TG KO mice) by mating PHGPx-loxP TG/KO mice with tissue-specific Cre expressing PHGPx heterozygous mice. In tissue specific PHGPx knockout mice, PHGPx-loxP transgene was specifically disrupted by tissue specific Cre recombinase.

Mentions: As shown in Fig. 6, we established floxed PHGPx mice (PHGPx-loxP TG/KO mice; control mice) using the transgenic complementation rescue method [32]. Transgenically rescued floxed mice exhibited normal growth and fertility. The expression of PHGPx protein in several tissues and the distribution of PHGPx in seminiferous tubules of testes was almost the same as in wild type mice. These results show that the approximately 5 kbp promoter regions in the PHGPx-loxP transgene are sufficient for normal regulation of PHGPx expression in embryogenesis and spermatogenesis in control mice. Our results also demonstrate that the transgenic complementation rescue method using this PHGPx-loxP transgene is useful for functional analysis of the three isoforms of PHGPx in mice by mutation of their start codons. The control floxed mice are useful for generating tissue-specific PHGPx conditional KO mice by mating these mice with tissue-specific Cre expressing PHGPx heterozygous mice. Because tissue specific Cre recombinase can disrupt the PHGPx-loxP transgene in PHGPx-loxP TG/KO mice, we could easily generate the tissue-specific PHGPx KO mice using this system (Fig. 6).


New Strategy of Functional Analysis of PHGPx Knockout Mice Model Using Transgenic Rescue Method and Cre-LoxP System.

Imai H - J Clin Biochem Nutr (2009)

Our established transgenic rescued floxed PHGPx knockout mice (Control mice) and tissue specific conditional PHGPx knockout mice by deletion of PHGPx-loxP transgene using Cre-loxP system. We established three lines of PHGPx-loxP TG/KO mice (TG KO, Control mice), all of which were viable and fertile, with body weights indistinguishable from those of wild-type mice. The PHGPx-loxP transgene could transcribe only PHGPx mRNA in the PHGPx-loxP TG/KO mice at nearly the same level as in the wild-type mice. Using a Cre-loxP conditional knockout strategy, we could generate tissue-specific PHGPx knockout mice (Cre TG KO mice) by mating PHGPx-loxP TG/KO mice with tissue-specific Cre expressing PHGPx heterozygous mice. In tissue specific PHGPx knockout mice, PHGPx-loxP transgene was specifically disrupted by tissue specific Cre recombinase.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2803127&req=5

Figure 6: Our established transgenic rescued floxed PHGPx knockout mice (Control mice) and tissue specific conditional PHGPx knockout mice by deletion of PHGPx-loxP transgene using Cre-loxP system. We established three lines of PHGPx-loxP TG/KO mice (TG KO, Control mice), all of which were viable and fertile, with body weights indistinguishable from those of wild-type mice. The PHGPx-loxP transgene could transcribe only PHGPx mRNA in the PHGPx-loxP TG/KO mice at nearly the same level as in the wild-type mice. Using a Cre-loxP conditional knockout strategy, we could generate tissue-specific PHGPx knockout mice (Cre TG KO mice) by mating PHGPx-loxP TG/KO mice with tissue-specific Cre expressing PHGPx heterozygous mice. In tissue specific PHGPx knockout mice, PHGPx-loxP transgene was specifically disrupted by tissue specific Cre recombinase.
Mentions: As shown in Fig. 6, we established floxed PHGPx mice (PHGPx-loxP TG/KO mice; control mice) using the transgenic complementation rescue method [32]. Transgenically rescued floxed mice exhibited normal growth and fertility. The expression of PHGPx protein in several tissues and the distribution of PHGPx in seminiferous tubules of testes was almost the same as in wild type mice. These results show that the approximately 5 kbp promoter regions in the PHGPx-loxP transgene are sufficient for normal regulation of PHGPx expression in embryogenesis and spermatogenesis in control mice. Our results also demonstrate that the transgenic complementation rescue method using this PHGPx-loxP transgene is useful for functional analysis of the three isoforms of PHGPx in mice by mutation of their start codons. The control floxed mice are useful for generating tissue-specific PHGPx conditional KO mice by mating these mice with tissue-specific Cre expressing PHGPx heterozygous mice. Because tissue specific Cre recombinase can disrupt the PHGPx-loxP transgene in PHGPx-loxP TG/KO mice, we could easily generate the tissue-specific PHGPx KO mice using this system (Fig. 6).

Bottom Line: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids.PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription.All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane Minato-ku Tokyo 108-8641, Japan.

ABSTRACT
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.

No MeSH data available.


Related in: MedlinePlus