Effects of Sperm Pretreatment on Efficiency of ICSI-Mediated Gene Transfer in Pigs
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概要
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Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, GFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further the improve rates of blastocyst formation and GFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and GFP expression that tended to be generally high (23.0%, 14/61 - 33.8%, 26/77 and 42.9%, 6/14 - 66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and GFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.
- 日本繁殖生物学会の論文
- 2007-12-01
著者
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UENO Satoshi
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji U
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NAGASHIMA Hiroshi
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji U
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TOMII Ryo
Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji Un
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KUROME Mayuko
Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji Un
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Ueno Satoshi
Lab. Of Developmental Engineering Dep. Of Life Sciences School Of Agriculture Meiji Univ.
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HIRUMA Katsumi
Laboratory of Developmental Engineering Department of Life Science, School of Agriculture, Meiji Uni
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SAITO Hitoshi
Laboratory of Developmental Engineering Department of Life Science, School of Agriculture, Meiji Uni
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Nagashima Hiroshi
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Tomii Ryo
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Ueno Satoshi
Laboratory Of Developmental Engineering Department Of Life Science Meiji University
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Kurome Mayuko
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Hiruma Katsumi
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Saitoh Hitoshi
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Nagashima Hiroshi
Lab. Of Developmental Engineering Dep. Of Life Sciences School Of Agriculture Meiji Univ.
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Nagashima Hiroshi
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Kurome Mayuko
Laboratory Of Developmental Engineering Department Of Life Science School Of Agriculture Meiji Unive
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Nagashima Hiroshi
Laboratory For Evolutionary Morphology Riken Cdb
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