利用CRISPR-Cas9技術(shù)構(gòu)建MSX1基因敲除人胚胎干細胞系
發(fā)布時間：2018-10-18 20:07
【摘要】：目的:1.采用CRISPR-Cas9基因編輯技術(shù),構(gòu)建與牙齒發(fā)育相關(guān)的MSX1基因敲除人胚胎干細胞(hESc)穩(wěn)定細胞系。2.研究MSX1基因敲除hESc系的多能性。3.探討MSX1基因敲除hESc的擬胚體(EB)向外、中、內(nèi)三胚層細胞分化的能力。方法:1.采用分子克隆的技術(shù)構(gòu)建pX330-MSX1KO-sgRNA載體、MSX1基因同源重組敲除載體(donor)。2.將pX330-MSX1KO-sgRNA載體以及donor共同電轉(zhuǎn)入hESc,PCR檢測和測序鑒定鑒定存活的克隆。3.對細胞進行形態(tài)學觀察、染色體核型分析、流式細胞術(shù),檢測MSX1基因雙敲除hESc(H1-MSX1-DKO)的多能性。4.體外形成H1-MSX1-DKO的擬胚體(EB),并誘導EB向外、中、內(nèi)三胚層細胞分化,收集EB分化前后(D0和D8)細胞的總RNA,采用實時熒光定量PCR法檢測各組細胞三胚層細胞標記物的表達情況。結(jié)果:1.質(zhì)粒測序結(jié)果顯示pX330-MSX1KO-sgRNA載體以及donor成功構(gòu)建,并成功獲得78株MSX1基因單敲除和2株MSX1基因雙敲除hESc系(H1-MSX1-SKO,H1-MSX1-DKO)。2.MSX1基因敲除hESc表現(xiàn)出典型的人胚胎干細胞的形態(tài);另外,細胞除了保持原有正常核型外,還高表達多能性標記物OCT4和SSEA4。3.實時熒光定量PCR結(jié)果顯示分化D8的細胞外、中、內(nèi)三胚層細胞標記物的表達水平都不同程度的高于未分化的細胞。結(jié)論:運用CRISPR-Cas9基因編輯技術(shù)能夠?qū)崿F(xiàn)對人胚胎干細胞的特定的編輯;MSX1基因敲除hESc還能保持干細胞的多能性并向三胚層細胞分化。我們獲得的MSX1基因敲除hESc可以用于進一步探討牙齒發(fā)育的分子機制,進而為人類研究MSX1基因異常導致的先天缺牙等相關(guān)疾病的治療提供一定參考。
[Abstract]:Objective: 1. CRISPR-Cas9 gene editing technique was used to construct MSX1 gene knockout human embryonic stem cell (hESc) stable cell line. 2. 2. To study the pluripotency of MSX1 knockout hESc lines. To study the ability of MSX1 knockout hESc embryoid (EB) to differentiate into outer, mesoderm and endoderm cells. Methods: 1. PX330-MSX1KO-sgRNA vector was constructed by molecular cloning and MSX1 gene homologous recombination knockout vector (donor). 2. Electroporation of pX330-MSX1KO-sgRNA vector and donor into hESc,PCR detection and sequencing to identify the surviving clones. 3. 3. Morphological observation, karyotype analysis and flow cytometry were used to detect the pluripotency of MSX1 double knockout hESc (H1-MSX1-DKO). 4. The embryoid (EB), of H1-MSX1-DKO was formed in vitro and induced the differentiation of EB to outer, mesoderm and endodermal cells. The total RNA, of EB cells before and after (D0 and D8) differentiation were collected to detect the expression of the markers of tridermal cells in each group by real-time fluorescence quantitative PCR method. The result is 1: 1. The results of plasmid sequencing showed that pX330-MSX1KO-sgRNA vector and donor were successfully constructed and 78 strains of MSX1 gene single knockout and 2 MSX1 gene double knockout hESc lines (H1-MSX1-SKOH1-MSX1-DKO) were successfully constructed. 2.MSX1 gene knockout hESc showed typical morphology of human embryonic stem cells. In addition to maintaining normal karyotypes, cells also expressed high expression of multipotent markers OCT4 and SSEA4.3.. The results of real-time fluorescence quantitative PCR showed that the expression levels of markers in differentiated D8 cells were higher than those in undifferentiated cells to some extent. Conclusion: the specific editing of human embryonic stem cells can be achieved by using CRISPR-Cas9 gene editing technique, and MSX1 knockout hESc can also maintain the pluripotency of stem cells and differentiate into tridermal cells. The MSX1 knockout hESc we obtained can be used to further explore the molecular mechanism of tooth development and provide a certain reference for the study of the treatment of congenital tooth defects caused by abnormal MSX1 gene.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R78
[Abstract]:Objective: 1. CRISPR-Cas9 gene editing technique was used to construct MSX1 gene knockout human embryonic stem cell (hESc) stable cell line. 2. 2. To study the pluripotency of MSX1 knockout hESc lines. To study the ability of MSX1 knockout hESc embryoid (EB) to differentiate into outer, mesoderm and endoderm cells. Methods: 1. PX330-MSX1KO-sgRNA vector was constructed by molecular cloning and MSX1 gene homologous recombination knockout vector (donor). 2. Electroporation of pX330-MSX1KO-sgRNA vector and donor into hESc,PCR detection and sequencing to identify the surviving clones. 3. 3. Morphological observation, karyotype analysis and flow cytometry were used to detect the pluripotency of MSX1 double knockout hESc (H1-MSX1-DKO). 4. The embryoid (EB), of H1-MSX1-DKO was formed in vitro and induced the differentiation of EB to outer, mesoderm and endodermal cells. The total RNA, of EB cells before and after (D0 and D8) differentiation were collected to detect the expression of the markers of tridermal cells in each group by real-time fluorescence quantitative PCR method. The result is 1: 1. The results of plasmid sequencing showed that pX330-MSX1KO-sgRNA vector and donor were successfully constructed and 78 strains of MSX1 gene single knockout and 2 MSX1 gene double knockout hESc lines (H1-MSX1-SKOH1-MSX1-DKO) were successfully constructed. 2.MSX1 gene knockout hESc showed typical morphology of human embryonic stem cells. In addition to maintaining normal karyotypes, cells also expressed high expression of multipotent markers OCT4 and SSEA4.3.. The results of real-time fluorescence quantitative PCR showed that the expression levels of markers in differentiated D8 cells were higher than those in undifferentiated cells to some extent. Conclusion: the specific editing of human embryonic stem cells can be achieved by using CRISPR-Cas9 gene editing technique, and MSX1 knockout hESc can also maintain the pluripotency of stem cells and differentiate into tridermal cells. The MSX1 knockout hESc we obtained can be used to further explore the molecular mechanism of tooth development and provide a certain reference for the study of the treatment of congenital tooth defects caused by abnormal MSX1 gene.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R78
【參考文獻】
相關(guān)期刊論文 前9條
1 朱姝;盛巍;陳旭;;與先天缺牙相關(guān)的全身綜合征[J];中國實用口腔科雜志;2016年09期
2 郭曉強;;CRISPR-Cas9技術(shù)發(fā)展史:25年的科學歷程[J];自然雜志;2016年04期
3 陳倩倩;李月;孟郁潔;戚曉鵬;;MSX1基因與非綜合征型唇/腭裂關(guān)聯(lián)研究進展[J];中國優(yōu)生與遺傳雜志;2015年12期
4 李聰;曹文廣;;CRISPR/Cas9介導的基因編輯技術(shù)研究進展[J];生物工程學報;2015年11期
5 劉志國;;CRISPR/Cas9系統(tǒng)介導基因組編輯的研究進展[J];畜牧獸醫(yī)學報;2014年10期
6 李藍;張博文;趙志河;;牙缺失基因的研究進展[J];華西口腔醫(yī)學雜志;2013年04期
7 潘爽;凌均h，
本文編號：2280228
本文鏈接：http://www.sikaile.net/kejilunwen/jiyingongcheng/2280228.html
最近更新
教材專著
