本文選題：精子載體法　切入點：轉(zhuǎn)基因動物　出處：《萊陽農(nóng)學(xué)院》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：精子介導(dǎo)基因轉(zhuǎn)移是以精子為載體,在受精時將外源目的基因?qū)肼涯讣毎?是目前轉(zhuǎn)基因動物研究中簡單而高效的方法學(xué)之一。本研究以ICR小白鼠為試驗動物,用精子載體法制備轉(zhuǎn)基因小鼠模型,并根據(jù)得到的結(jié)果分析討論精子載體法制備轉(zhuǎn)基因動物的可行性及優(yōu)缺點。 本文進行了如下研究: 外源DNA轉(zhuǎn)染小鼠精子的研究:利用DIG末端標(biāo)記技術(shù)和免疫組化技術(shù)研究小鼠精子轉(zhuǎn)染外源DNA的效率。試驗結(jié)果發(fā)現(xiàn),不同小鼠個體精子轉(zhuǎn)染陽性率有明顯差異,平均為13%左右。小鼠精子具有自發(fā)結(jié)合外源DNA能力,結(jié)合部位集中在精子頂體后軀,結(jié)合的外源DNA首先“掛”在精子膜表面,繼而被內(nèi)化轉(zhuǎn)運到細胞內(nèi),但并非所有的結(jié)合DNA都被內(nèi)化。 精子介導(dǎo)法制備轉(zhuǎn)基因小鼠的試驗研究:利用考馬斯亮藍染色技術(shù)評價小鼠精子頂體反應(yīng)發(fā)生的情況,選擇合適的體外受精液。利用小鼠體外受精技術(shù),將體外轉(zhuǎn)染GFP并獲能的小鼠精子對成熟卵母細胞進行體外受精,受精卵進行體外培養(yǎng),熒光顯微鏡下觀察胚胎,結(jié)果表達GFP的陽性率為4.7%。試驗結(jié)果證明了精子介導(dǎo)法制備轉(zhuǎn)基因小鼠的可行性。 乙肝病毒結(jié)合蛋白表達載體的構(gòu)建:利用PCR和基因重組技術(shù)構(gòu)建了HBV結(jié)合蛋白表達載體,經(jīng)過酶切鑒定和基因測序證實了載體序列的正確性,并作為外源DNA準備用于轉(zhuǎn)染小鼠精子制備動物疾病模型。 本研究初步建立了利用精子載體法制備轉(zhuǎn)基因小鼠的平臺,為今后制備動物疾病模型奠定了基礎(chǔ)。但沒有進行HBV結(jié)合蛋白基因的轉(zhuǎn)染,今后還需對本試驗作進一步的研究。
[Abstract]:Sperm mediated gene transfer to spermatozoa during fertilization, the exogenous gene into oocytes, transgenic animal research is currently one of the simple and efficient methodology. In this study, ICR mice as experimental animal, transgenic mice model using sperm vector method, and discussed the feasibility and advantages and disadvantages sperm carrier preparation of transgenic animal according to the results.
The following study is carried out in this paper.
Study of exogenous DNA transfection of mouse spermatozoa: efficiency by DIG end labeling and immunohistochemistry study of mouse spermatozoa transfected with exogenous DNA. The test results show that the obvious difference between the positive rate of different individual mouse sperm transfection, the average is about 13%. The mouse sperm has spontaneous binding of exogenous DNA, the binding site focused on sperm acrosome the hindquarters, exogenous DNA with first "hang" on the surface of the sperm membrane, then transport is internalized into cells, but not all of the combination of DNA have been internalized.
Study on Preparation of transgenic mouse sperm mediated method: Using Coomassie brilliant blue staining technique to evaluate mouse sperm acrosome reaction, select the appropriate in vitro fertilization in vitro. The semen of mice, the mice sperm in vitro transfection of GFP and capacitation in vitro fertilization of mature oocytes, fertilized eggs were cultured in vitro. The embryo was observed under fluorescence microscope, the positive expression rate of GFP proved the feasibility of preparation of transgenic mouse sperm mediated by 4.7%. test results.
Hepatitis B virus binding protein expression vector construction: to construct the HBV binding protein expression vector using PCR and gene recombination technique. After enzyme digestion and sequencing confirmed the correctness of the vector sequence, and as exogenous DNA prepared for transfection of mouse sperm preparation of animal disease model.
In this study, we initially established a platform for preparing transgenic mice by sperm carrier method, which laid the foundation for the preparation of animal disease models. However, no HBV binding protein gene was transfected. Further research is needed in the future.

【學(xué)位授予單位】：萊陽農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R-332

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 嵇斌;睪丸內(nèi)注射法建立轉(zhuǎn)基因小鼠的實驗研究[D];東北農(nóng)業(yè)大學(xué);2008年

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本文編號：1565508