發(fā)布時(shí)間：2018-05-05 15:26

  本文選題：基因組編輯 + 耳成纖維細(xì)胞�。� 參考：《上海海洋大學(xué)》2015年碩士論文

【摘要】：動(dòng)物基因組編輯技術(shù)是指研究人員能夠依照特定的目的去修飾動(dòng)物的遺傳組成,這是一項(xiàng)綜合性的并且富有挑戰(zhàn)性的技術(shù)。該技術(shù)由于能夠比較容易并且精確的對(duì)基因組進(jìn)行DNA的添加、刪除和置換的操作,使得其在改良畜產(chǎn)品質(zhì)量,提高生產(chǎn)性能,研究疾病模型,研發(fā)生物醫(yī)藥產(chǎn)品等方面都顯示出廣闊的應(yīng)用前景�；蚨c(diǎn)敲入時(shí),需要為一些外源基因,如報(bào)告基因、自殺基因、篩選基因、治療性基因和外源性?xún)?yōu)良性狀基因提供一個(gè)安全可靠的停泊“港口”,在這些“港口”外源基因既可穩(wěn)定高效表達(dá),又對(duì)細(xì)胞和組織無(wú)已知的副作用。近年來(lái),轉(zhuǎn)基因動(dòng)物的生物安全性一直是人們討論的熱點(diǎn)問(wèn)題。從目前已經(jīng)已發(fā)表的文獻(xiàn)看來(lái),針對(duì)豬基因組安全港位點(diǎn)的研究鳳毛麟角。因此,尋找合適的安全港位點(diǎn)對(duì)于轉(zhuǎn)基因豬的制備顯得尤為重要。在克隆動(dòng)物的制備中,耳成纖維細(xì)胞作為供核體細(xì)胞具有取材簡(jiǎn)便,成本低,對(duì)動(dòng)物生長(zhǎng)影響小等優(yōu)點(diǎn),并且從耳部組織的采樣能夠?qū)崿F(xiàn)活體取樣,從而為優(yōu)良豬種的保種育種奠定基礎(chǔ)。本研究第一部分采用組織塊培養(yǎng)法分離、培養(yǎng)并建立豬耳成纖維細(xì)胞系,然后用電轉(zhuǎn)染的方法將質(zhì)粒p EGFP-C1導(dǎo)入該細(xì)胞系,探索并優(yōu)化其最適條件和參數(shù)。最后成功建立上海白豬耳成纖維細(xì)胞系。電轉(zhuǎn)后經(jīng)過(guò)對(duì)各組轉(zhuǎn)染效果的觀察,發(fā)現(xiàn)在DNA量為20 ng,電壓為150 V,電阻為500Ω,電容為500μF的條件下進(jìn)行電轉(zhuǎn),并且電轉(zhuǎn)后經(jīng)37℃孵育,其效果最好。第二部分為了研究CCR5基因作為安全港基因的可能性,首先分析了CCR5基因序列,發(fā)現(xiàn)該基因位于豬基因組第13號(hào)染色體上,基因總長(zhǎng)4 565 bp,CDS區(qū)位于第二外顯子上。然后針對(duì)CDS區(qū)atg下游約110 bp處分別設(shè)計(jì)并成功構(gòu)建了TALEN和CRISPR/Cas9系統(tǒng)的打靶載體,并將其分別電轉(zhuǎn)染豬耳成纖維細(xì)胞。然后用Cruise TM酶對(duì)細(xì)胞池進(jìn)行酶切驗(yàn)證后,發(fā)現(xiàn)TALEN打靶CCR5基因CDS區(qū)的結(jié)果為陰性,CRISPR的pool驗(yàn)證顯示敲除效率很低。初步懷疑這兩種技術(shù)在該位點(diǎn)上進(jìn)行打靶可能存在的脫靶效應(yīng)。第三部分為檢測(cè)分析上海梅山豬封閉群攜帶豬內(nèi)源性逆轉(zhuǎn)錄病毒(porcine endogenous retrovirus,PERV)存在與表達(dá)的情況,為該豬種作為器官移植供體的生物安全性提供現(xiàn)實(shí)依據(jù),筆者從上海嘉定區(qū)梅山豬育種中心的梅山豬封閉群內(nèi)隨機(jī)采集45頭豬的耳組織,并構(gòu)建耳成纖維細(xì)胞系,利用PCR檢測(cè)PERV前病毒核心蛋白基因(gag)、多聚酶基因(pol)以及囊膜基因(env)的基因組DNA,利用RT-PCR的方法檢測(cè)m RNA表達(dá)狀況。結(jié)果發(fā)現(xiàn)被檢測(cè)的45份樣品均攜帶完整的3種亞型的PERV前病毒DNA;RT-PCR結(jié)果顯示,45份樣品中有40份同時(shí)有PERV-A和PERV-B兩種亞型的表達(dá),還有5份樣品僅檢測(cè)到PERV-B亞型的表達(dá),未檢測(cè)到PERV-C亞型的表達(dá)。鑒于生物安全性方面的考慮,該豬種能否作為人異種移植的供體仍有待進(jìn)一步商榷。
[Abstract]:Animal genome editing is a comprehensive and challenging technique that allows researchers to modify the genetic composition of animals for specific purposes. This technology can easily and accurately add, delete and replace DNA to the genome, so that it can improve the quality of animal products, improve the production performance, and study the disease model. Research and development of biomedical products and other aspects have shown a broad application prospects. When gene knockout, we need to provide a safe and reliable berthing "port" for some foreign genes, such as reporter gene, suicide gene, screening gene, therapeutic gene and exogenous good trait gene. In these ports, exogenous genes can be expressed stably and efficiently without known side effects on cells and tissues. In recent years, the biological safety of transgenic animals has been a hot issue. According to the published literature, few studies have been done on the safe harbour site of pig genome. Therefore, it is very important to find a suitable safe port for the preparation of transgenic pigs. In the preparation of cloned animals, ear fibroblasts as nuclear donor cells have the advantages of simple selection, low cost and little influence on animal growth. So as to lay a foundation for the conservation breeding of superior pig breeds. In the first part of the study, the fibroblast cell line of porcine ear was isolated and established by tissue mass culture method. Then the plasmid p EGFP-C1 was transfected into the cell line by electrotransfection, and the optimum conditions and parameters were explored and optimized. Finally, Shanghai White Pig ear fibroblast cell line was successfully established. After electrotransposing, we observed the transfection effect of each group, and found that under the condition of DNA 20 ng, voltage 150 V, resistance 500 惟 and capacitance 500 渭 F, the best effect was obtained after incubation at 37 鈩，

本文編號(hào)：1848218