CRISPR-Cas9介導(dǎo)FGF5敲除及Noggin過表達(dá)對毛發(fā)生長相關(guān)因子影響
發(fā)布時間：2018-09-18 13:45
【摘要】：基因編輯技術(shù),興起于上世紀(jì)80年代,開啟了人1門對基因功能研究的熱潮。CRISPR-Cas9作為近年來新興的基因編輯技術(shù),具有載體構(gòu)建簡單、基因敲除率高等優(yōu)勢。絨山羊作為我國獨(dú)特的生物資源,具有產(chǎn)絨量高,絨質(zhì)好等諸多優(yōu)點(diǎn),其羊絨更具有“生物黃金”的美譽(yù)。因此進(jìn)行高產(chǎn)絨量的品種選育具有極高的意義和巨大的市場潛力。而毛發(fā)生長涉及到一個及其復(fù)雜的基因調(diào)控機(jī)制。以往研究表明,Noggin基因的過表達(dá)和FGF5基因的敲除都會促進(jìn)毛發(fā)生長。本實(shí)驗(yàn)主要針對毛發(fā)生長正調(diào)控基因Noggin和負(fù)調(diào)控基因FGF5,利用CRISPR-Cas9技術(shù)和基因隨機(jī)整合技術(shù),研究CRISPR-Cas9的敲除效率及FGF5敲除Noggin基因過表達(dá)同時作用后,在轉(zhuǎn)錄水平上檢測其對毛發(fā)生長相關(guān)基因的mRNA表達(dá)量,從而預(yù)測FGF5、Noggin雙基因編輯對毛發(fā)生長的影響。1、小鼠胎兒成纖維細(xì)胞轉(zhuǎn)染條件優(yōu)化本實(shí)驗(yàn)利用電轉(zhuǎn)染法,以pCMV-DsRed質(zhì)粒質(zhì)量梯度和電轉(zhuǎn)染電壓和脈沖時間組合參數(shù)梯度,研究了轉(zhuǎn)染小鼠胎兒成纖維細(xì)胞的最佳轉(zhuǎn)染條件,結(jié)果顯示電轉(zhuǎn)染質(zhì)�？傎|(zhì)量、電壓、脈沖時間的最佳組合為10μg/160V/5mM。2、gRNA-FGF5、Noggin隨機(jī)整合載體pK14-Noggin和Noggin定點(diǎn)整合載體pKI-Noggin的構(gòu)建本研究成功的構(gòu)建了針對小鼠FGF5基因的向?qū)NA(gRNA-FGF5),結(jié)果顯示其可成功引導(dǎo)Cas9蛋白到目的靶位點(diǎn)進(jìn)行雙鏈DNA切割。本實(shí)驗(yàn)同時成功構(gòu)建了兩個Noggin基因皮膚特異性表達(dá)載體。一個為Noggin隨機(jī)整合載體pK14-Noggin,用于Noggin過表達(dá)研究。另一個為Noggin定點(diǎn)整合載體pKI-Noggin,用于今后CRISPR/cas9,gRNA-FGF5介導(dǎo)Noggin定點(diǎn)整合模式動物構(gòu)建。3、FGF5基因敲除效率和Noggin基因隨機(jī)整合檢測我們對以上實(shí)驗(yàn)構(gòu)建的gRNA-FGF5和Noggin基因皮膚特異性隨機(jī)整合載體pK14-Noggin進(jìn)行效率檢測。首先,我們通過電轉(zhuǎn)染的方法將Cas9質(zhì)粒與gRNA-FGF5共同轉(zhuǎn)染小鼠胎兒成纖維細(xì)胞,提取細(xì)胞總基因組作為模板,用FGF5靶位點(diǎn)檢測引物進(jìn)行PCR反應(yīng),并利用Surveyor突變檢測試劑盒檢測,結(jié)果證明實(shí)驗(yàn)設(shè)計(jì)的gRNA-FGF5可以高效的引導(dǎo)Cas9蛋白對目標(biāo)靶點(diǎn)的切割。本研究還運(yùn)用T-A克隆的方法將PCR產(chǎn)物連入pMD19-T載體,隨機(jī)挑取20個陽性重組子委托北京華大基因公司進(jìn)行測序。結(jié)果顯示：隨機(jī)挑取的20個重組子中有11個在靶位點(diǎn)具有不同程度的堿基缺失與插入,由此結(jié)論獲得突變率約為55%。其次,我們將pK14-Noggin質(zhì)粒電轉(zhuǎn)染小鼠胎兒成纖維細(xì)胞,48h后,提取細(xì)胞總蛋白,利用western blot檢測,結(jié)果顯示Noggin蛋白相對表達(dá)量是相同來源及代次野生型對照細(xì)胞的1.41倍。4、FGF5敲除Noggin過表達(dá)對毛發(fā)生長相關(guān)因子的影響本實(shí)驗(yàn)將Cas9、gRNA-FGF5轉(zhuǎn)染MEF細(xì)胞得到EG1; pK14-Noggin轉(zhuǎn)染小鼠MEF細(xì)胞得到EG2; Cas9、gRNA-FGF5、pK14-Noggin共轉(zhuǎn)染小鼠MEF細(xì)胞得到EG3,并以未轉(zhuǎn)基因的小鼠MEF細(xì)胞CG作為對照。轉(zhuǎn)染48h后,以4種細(xì)胞的總cDNA作為模板,利用qPCR的方法,在mRNA水平上檢測Tβ4、β-catenin、 vegf三個基因的mRNA相對表達(dá)量,結(jié)果顯示：FGF5敲除的細(xì)胞β-catenin、 Vegf、Tβ4mRNA相對表達(dá)量分別是空白對照的2.82/1.37/1.47倍。noggin隨機(jī)整合的細(xì)胞β-catenin、Vegf、Tβ4 mRNA相對表達(dá)量分別是空白對照的2.44/1.32/1.37倍 。FGF5敲除、noggin隨機(jī)整合的細(xì)胞β-catenin、 Vegf、Tβ4 mRNA相對表達(dá)量分別是空白對照的3.22/1.69/1.71倍。
[Abstract]:Gene editing technology, rising in the 1980s, has opened a new upsurge in gene function research. As a new gene editing technology in recent years, CRISPR-Cas9 has the advantages of simple vector construction and high gene knockout rate. As a unique biological resource in China, cashmere goats have many advantages, such as high cashmere yield, good cashmere quality and so on. Therefore, it is of great significance and great market potential to breed varieties with high cashmere yield. Hair growth involves a complicated gene regulation mechanism. Previous studies have shown that overexpression of Noggin gene and knockout of FGF5 gene can promote hair growth. The knockout efficiency of CRISPR-Cas9 and the over-expression of Noggin gene knocked out by FGF5 were studied by using CRISPR-Cas9 technique and random integration technique. The mRNA expression of genes related to hair growth was detected at transcriptional level to predict the editorial pairs of FGF5 and Noggin genes. Effects of Hair Growth on the Transfection Conditions of Mouse Fetal Fibroblasts The optimal combination was 10 ug/160V/5mM.2, gRNA-FGF5, Noggin random integration vector pK14-Noggin and Noggin site-directed integration vector pKI-Noggin. In this study, we successfully constructed a guided RNA (gRNA-FGF5) targeting mouse fibroblast growth factor 5 gene. The results showed that it could successfully guide Cas9 protein to target sites for double stranded DNA cleavage. Two Noggin gene skin-specific expression vectors were successfully constructed. One was a Noggin random integration vector pK14-Noggin for Noggin overexpression study. The other was a Noggin site-specific integration vector pKI-Noggin for future CRISPR/cas9. GRNA-FGF5 mediated Noggin site-specific integration model animal construction. The efficiency of gRNA-FGF5 and Noggin gene skin-specific random integrative vector pK14-Noggin constructed in the above experiments was tested by machine integration test. First, we transfected the cas9 plasmid and gRNA-FGF5 into mouse fetal fibroblasts by electroporation, extracted the total genome of the cells as a template, and detected the primers with the target site of FGF5. The results of PCR reaction and Surveyor mutation detection kit showed that the designed gRNA-FGF5 could efficiently guide the cleavage of Cas9 protein to target sites. In this study, the PCR products were linked to pMD19-T vector by T-A cloning, and 20 positive recombinants were randomly selected for sequencing by Beijing Huada Gene Company. The results showed that 11 of the 20 recombinants were deleted and inserted at different levels at the target site, and the mutation rate was about 55%. Secondly, we transfected pK14-Noggin plasmid into mouse fetal fibroblasts. After 48 hours, the total cell protein was extracted and detected by Western blot. The results showed that Noggin protein was relative. In this study, EG1 was obtained by transfecting Cas9, gRNA-FGF5 into MEF cells, EG2 was obtained by transfecting pK14-Noggin into MEF cells, EG3 was obtained by co-transfecting mouse MEF cells with Cas9, gRNA-FGF5 and pK14-Noggin, and EG3 was obtained by co-transfecting mouse MEF cells with pK14-Noggin. After 48 hours of transfection, the relative mRNA expression levels of T-beta-4, beta-catenin and VEGF genes were detected by q-PCR. The results showed that the relative mRNA expression levels of beta-catenin, Vegf and T-beta-4 in the knockout cells were 2.82/1.37/1.47 times higher than those in the blank control. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 2.44/1.32/1.37 times higher than that in blank control cells, respectively. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 3.22/1.69/1.71 times higher than that in blank control cells.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.2
本文編號：2248105
[Abstract]:Gene editing technology, rising in the 1980s, has opened a new upsurge in gene function research. As a new gene editing technology in recent years, CRISPR-Cas9 has the advantages of simple vector construction and high gene knockout rate. As a unique biological resource in China, cashmere goats have many advantages, such as high cashmere yield, good cashmere quality and so on. Therefore, it is of great significance and great market potential to breed varieties with high cashmere yield. Hair growth involves a complicated gene regulation mechanism. Previous studies have shown that overexpression of Noggin gene and knockout of FGF5 gene can promote hair growth. The knockout efficiency of CRISPR-Cas9 and the over-expression of Noggin gene knocked out by FGF5 were studied by using CRISPR-Cas9 technique and random integration technique. The mRNA expression of genes related to hair growth was detected at transcriptional level to predict the editorial pairs of FGF5 and Noggin genes. Effects of Hair Growth on the Transfection Conditions of Mouse Fetal Fibroblasts The optimal combination was 10 ug/160V/5mM.2, gRNA-FGF5, Noggin random integration vector pK14-Noggin and Noggin site-directed integration vector pKI-Noggin. In this study, we successfully constructed a guided RNA (gRNA-FGF5) targeting mouse fibroblast growth factor 5 gene. The results showed that it could successfully guide Cas9 protein to target sites for double stranded DNA cleavage. Two Noggin gene skin-specific expression vectors were successfully constructed. One was a Noggin random integration vector pK14-Noggin for Noggin overexpression study. The other was a Noggin site-specific integration vector pKI-Noggin for future CRISPR/cas9. GRNA-FGF5 mediated Noggin site-specific integration model animal construction. The efficiency of gRNA-FGF5 and Noggin gene skin-specific random integrative vector pK14-Noggin constructed in the above experiments was tested by machine integration test. First, we transfected the cas9 plasmid and gRNA-FGF5 into mouse fetal fibroblasts by electroporation, extracted the total genome of the cells as a template, and detected the primers with the target site of FGF5. The results of PCR reaction and Surveyor mutation detection kit showed that the designed gRNA-FGF5 could efficiently guide the cleavage of Cas9 protein to target sites. In this study, the PCR products were linked to pMD19-T vector by T-A cloning, and 20 positive recombinants were randomly selected for sequencing by Beijing Huada Gene Company. The results showed that 11 of the 20 recombinants were deleted and inserted at different levels at the target site, and the mutation rate was about 55%. Secondly, we transfected pK14-Noggin plasmid into mouse fetal fibroblasts. After 48 hours, the total cell protein was extracted and detected by Western blot. The results showed that Noggin protein was relative. In this study, EG1 was obtained by transfecting Cas9, gRNA-FGF5 into MEF cells, EG2 was obtained by transfecting pK14-Noggin into MEF cells, EG3 was obtained by co-transfecting mouse MEF cells with Cas9, gRNA-FGF5 and pK14-Noggin, and EG3 was obtained by co-transfecting mouse MEF cells with pK14-Noggin. After 48 hours of transfection, the relative mRNA expression levels of T-beta-4, beta-catenin and VEGF genes were detected by q-PCR. The results showed that the relative mRNA expression levels of beta-catenin, Vegf and T-beta-4 in the knockout cells were 2.82/1.37/1.47 times higher than those in the blank control. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 2.44/1.32/1.37 times higher than that in blank control cells, respectively. The relative expression of beta-catenin, Vegf and Tbeta-4 mRNA in noggin-randomly integrated cells was 3.22/1.69/1.71 times higher than that in blank control cells.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.2
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