本文選題：精子載體法 + 脂質(zhì)體��； 參考：《河北農(nóng)業(yè)大學(xué)》2011年碩士論文

【摘要】：精子介導(dǎo)的基因轉(zhuǎn)移是指以精子作為外源基因的載體,在受精時(shí)將外源基因?qū)肼涯讣?xì)胞,使受精卵中含有外源基因,是目前轉(zhuǎn)基因動(dòng)物研究中簡(jiǎn)單而高效的方法之一。脂質(zhì)體可用于轉(zhuǎn)基因的研究,利用脂質(zhì)體可以和細(xì)胞膜融合的特點(diǎn),將外源基因送入細(xì)胞內(nèi)部。本實(shí)驗(yàn)利用脂質(zhì)體介導(dǎo)精子轉(zhuǎn)染的方法,研究了精子在介導(dǎo)基因轉(zhuǎn)移過程中精子與外源基因結(jié)合的影響因素,并對(duì)精子載體法制備FecB轉(zhuǎn)基因太行山羊進(jìn)行了初步研究。試驗(yàn)結(jié)果如下: 1.精子充分離心洗滌后,再與脂質(zhì)體-DNA復(fù)合物混合,可顯著提高脂質(zhì)體介導(dǎo)精子轉(zhuǎn)染外源DNA的效率。DNaseI消化前,洗滌組轉(zhuǎn)染精子陽性率為38%,而未處理精液組精子陽性率為14%;DNaseI消化后,洗滌組轉(zhuǎn)染精子陽性率為21%,而未處理精液組精子陽性率為5%,差異極顯著(P 0.01)。 2.脂質(zhì)體介導(dǎo)法可使外源DNA透過細(xì)胞膜內(nèi)化進(jìn)入精子胞質(zhì)或胞核內(nèi),精子轉(zhuǎn)染外源DNA用DNaseI消化后能檢測(cè)到精子中外源DNA的存在。太行山羊精子與外源DNA混合培養(yǎng)后,用熒光原位雜交檢測(cè)到18.3%的精子結(jié)合有外源DNA,脂質(zhì)體Lipofectamine2000介導(dǎo)后比率增加至38.7%,兩法的統(tǒng)計(jì)學(xué)分析存在顯著性差異(P0.01)。 3.利用共培養(yǎng)法轉(zhuǎn)染外源DNA,用DNaseI消化后,能檢測(cè)到精子中外源DNA的存在,山羊精子具有自發(fā)的結(jié)合外源DNA的能力。 4.對(duì)18只太行山羊母羊進(jìn)行誘導(dǎo)發(fā)情處理,精子與FecB基因共孵育后,對(duì)發(fā)情母羊進(jìn)行輸精,受孕率為51%,共產(chǎn)生后代12只,經(jīng)過PCR檢測(cè),未發(fā)現(xiàn)陽性后代。
[Abstract]:Spermatozoa mediated gene transfer refers to the introduction of exogenous genes into oocytes during fertilization, which is one of the simple and efficient methods in the research of transgenic animals. Liposomes can be used in the study of transgene, and foreign genes can be transferred into the cells by the fusion of liposome and cell membrane. In this study, the factors affecting the binding of sperm to foreign genes in the process of sperm mediated gene transfer were studied by using liposome-mediated sperm transfection, and the preparation of FecB transgenic Taihang Sheep by sperm carrier method was studied preliminarily. The results are as follows: 1. After full centrifugation, spermatozoa were mixed with liposome-DNA complex. The efficiency of transfection of exogenous DNA into sperm mediated by liposome. DNase I was significantly improved. The positive rate of sperm transfection in washing group was 38 and that in untreated semen group was 14% after DNase I digestion. The positive rate of sperm transfection in washing group was 21 and that in untreated semen group was 5. The difference was significant (P 0.01). 2. Liposome-mediated method can make exogenous DNA enter the sperm cytoplasm or nucleus through the cell membrane internalization. After the sperm transfection of exogenous DNA is digested with DNaseI, the presence of DNaseI can be detected in the spermatozoa. After mixed culture of sperm and exogenous DNA in Taihang Mountain sheep, 18.3% of spermatozoa combined with exogenous DNA was detected by fluorescence in situ hybridization, and the ratio of liposome Lipofectamine2000 mediated was increased to 38.7%. There was a significant difference between the two methods in statistical analysis (P 0.01). 3. Exogenous DNA could be detected by co-culture and digested with DNaseI. Goat spermatozoa had the ability to combine exogenous DNA spontaneously. 4. After the estrus induction treatment was carried out in 18 estrous sheep of Taihang Mountain sheep, the estrus estrus was inseminated by sperm and FecB gene. The pregnancy rate was 51%, and 12 offspring were produced. After PCR test, no positive offspring were found.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：S827

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