遺伝子異常に関わる対策 : 精子遺伝子導入による運動率と受精能の変化

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7週齢のB6D2F1雌雄マウス,同週齢のICR雌雄マウスを用い,生殖領域における遺伝子導入の臨床的問題点・安全性についてリポフェクション法によるマウス精子の遺伝子導入を行い検討した.精子運動率はDNAを含まないリポフェクション試薬に比べ,DNA濃度依存性に低下し,0.5μg/ml異常では統計学的有意差を認めた.IVFにおける受精率はDNAを加えることによりDNA濃度依存性に有意に低下した.精子への遺伝子導入は比較的簡便に行われると思われた.また遺伝子が導入された精子は卵子との受精が可能で外来性遺伝子が受精卵に伝達可能であることが証明された.一方で遺伝子導入により精子の一部に何らかの障害が起こり,精子の運動能や受精率の低下が起こると推察された.今後DNA導入精子の分別法の開発と効率かつ安全なDNA条件の設定が必要と考えられたTo clarify the problems of gene transfer in the fertility clinic, we investigated the safety and effectiveness of the gene transfer of mouse sperm using the lipofection method. We used the pCAGGS-lacZ plasmid connected with cytomegarovirus enhancer/chicken beta-actin promoter as an adventitious gene. After incubation of the DNA-liposome complex and mouse sperm of epididymis in the medium, we investigated the sperm motility after gene transfer, in vitro fertilization rate of the gene transferred sperm, and gene expression of the fertilized ovum and fetus. Sperm motility was lowered significantly with the increase in DNA density. The fertilization rate for in vitro fertilization was lowered significantly with the increase in DNA density. Gene expression was observed in some of the fertilized ova. In the fetus 12.5 days after embryo transfer, gene transfer was confirmed by the PCR method, but we could not find gene expression by X-gal staining. We proved that the sperm with a gene transferred by the lipofection method could fertilize the ovum and transfer adventitious gene to ova. We speculate that sperm motility and fertility rate were lowered because the introduction of DNA and the DNA-liposome complex destroyed parts of the cell. Development of a sperm fractionation method for DNA transfer and establishment of a method for effective and safe DNA transfer are awaited.

遺伝子異常に関わる対策 : 精子遺伝子導入による運動率と受精能の変化

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