遺伝子工学の微細操作法を利用した着床前受精卵の遺伝子診断および胚移植に関する研究

利用基因工程显微操作方法进行植入前受精卵及胚胎移植的基因诊断研究

• 批准号: 11770966
• 负责人: 具 然和
• 金额: $ 1.41万
• 依托单位: Suzuka University of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Young Scientists (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11770966/
• 关键词: 着床前期; 遺伝子構造; 人工受精; Single stranded conformational polymorphism; Denaturing gredient gel electrophoresis; Squeezing method; FISH; PCR; aspiration of blastomere; partial zona dissection

本研究では、着床前期の受精卵の単一遺伝子異常による遺伝疾患および染色体異常による奇形を検討した。2細胞期から8細胞期(桑実胚)の細胞を取りだし、細胞遺伝学的調査、蛍光染色による卵染色体の観察(FISH法)、遺伝子構造の観察(PCR法:single cell PCR,multiplex PCR)などによる着床前の遺伝子診断を行った。また、受精後、48時間〜72時間に卵管を貫流し、受精卵細胞を採取した。その後、modified Krebs-Ringers Bicarbonate(m-KRB)培養液に集め、90分間培養した後、顕微鏡上で卵細胞検査操作を行った。また、発情期にある雌マウスの卵管を貫流し、卵細胞を採取した。その後、以上と同じ方法で、卵を集めた。そして、雄マウスから採取した精子で人工受精を行った。その後、受精18日後に解剖し、受精率を確認した。その結果25%の確立で受精が成立した。また、採取した受精卵細胞の単一受精卵細胞のDNA量は、非常に微量のため、PCRは困難である。従って、Primer extension preamplification(PEP)を通じて全ての遺伝子を一次増幅した後、SSCP(Single stranded conformational polymorphism)およびDGGE(Denaturing gredient gel electrophoresis)を通じて遺伝疾患を検定した。しかし、突然変異がある場合は、dideoxytermiantion方法を利用したcyclic sequencing方法で突然変異が起こった部位の塩基配列を確認した。FISH法による受精卵細胞の検査は、人工受精卵を約48〜72時間培養し、4〜8細胞の卵細胞の内、二個の卵細胞を取りだし、RNaseの処理後、脱水し、常温で乾燥させた。体外受精された人工受精卵を約2〜3日培養し、4〜8細胞における卵細胞から単一細胞を取りだし、PCRおよびSSCPを正確に行った。そして、stock SolutionでHybridization Bufferを作る。その後、蛍光染色過程に従って染色を行った。また、MItotic Index,Pyknosis,Micronucliなどを観察した。

In this study, the zygote was fertilized in the preimplantation period, the zygote was often found in the zygote, the disease, the chromosome, the odd shape and the abnormal shape. (2) during the 8-cell stage (morula), the cells were collected and examined by light staining (FISH method), light staining (FISH method) and preimplantation (PCR method: single cell PCR,multiplex PCR). Oviduct abortion occurred during 48 to 72 hours after fertilization, and the fertilized eggs were harvested from the cells after fertilization. After incubation, the modified Krebs-Ringers Bicarbonate (m-KRB) culture solution was collected, and after 90 minutes of incubation, the oocyte culture operation was completed. Estrus, oestrus, oocytes, oocytes. After the treatment, the above methods are the same as those of the egg collection. The sperm were collected by artificial insemination and artificial insemination. The rats were dissected and the fertilization rate was confirmed after fertilization on the 18th day. The results showed that 25% of the patients were confirmed to be fertilized. The amount of DNA in the cell of a fertilized egg, the amount of PCR in the cell of a fertilized egg, and the amount of DNA in the cell of a fertilized egg. After SSCP (Single stranded conformational polymorphism), DGGE (Denaturing gredient gel electrophoresis), and Primer extension preamplification (PEP), all the patients were diagnosed with diseases and diseases. All of a sudden, all of a sudden, the dideoxytermiantion method makes use of the cyclic sequencing method to suddenly start the location, the base column and the confirmation column. The fertilized eggs were incubated in 4872 hours, 48-72 hours, 48-72 hours, 48-72 In vitro fertilization (IVF), the artificial fertilized eggs were cultured for 2 ~ 3 days, the oocytes were harvested in 4 ~ 8 cells, the eggs were collected in one cell, and the SSCP was correctly performed in PCR. Make a mistake, stock Solution the Hybridization Buffer to do the job. After the treatment, the light dyeing process was used to determine the dyeing process. Please, MItotic Index,Pyknosis,Micronucli please observe the situation.