利用CRISPR-Cas9技术构建MSX1基因敲除人胚胎干细胞系
发布时间：2018-10-18 20:07
【摘要】：目的:1.采用CRISPR-Cas9基因编辑技术,构建与牙齿发育相关的MSX1基因敲除人胚胎干细胞(hESc)稳定细胞系。2.研究MSX1基因敲除hESc系的多能性。3.探讨MSX1基因敲除hESc的拟胚体(EB)向外、中、内三胚层细胞分化的能力。方法:1.采用分子克隆的技术构建pX330-MSX1KO-sgRNA载体、MSX1基因同源重组敲除载体(donor)。2.将pX330-MSX1KO-sgRNA载体以及donor共同电转入hESc,PCR检测和测序鉴定鉴定存活的克隆。3.对细胞进行形态学观察、染色体核型分析、流式细胞术,检测MSX1基因双敲除hESc(H1-MSX1-DKO)的多能性。4.体外形成H1-MSX1-DKO的拟胚体(EB),并诱导EB向外、中、内三胚层细胞分化,收集EB分化前后(D0和D8)细胞的总RNA,采用实时荧光定量PCR法检测各组细胞三胚层细胞标记物的表达情况。结果:1.质粒测序结果显示pX330-MSX1KO-sgRNA载体以及donor成功构建,并成功获得78株MSX1基因单敲除和2株MSX1基因双敲除hESc系(H1-MSX1-SKO,H1-MSX1-DKO)。2.MSX1基因敲除hESc表现出典型的人胚胎干细胞的形态;另外,细胞除了保持原有正常核型外,还高表达多能性标记物OCT4和SSEA4。3.实时荧光定量PCR结果显示分化D8的细胞外、中、内三胚层细胞标记物的表达水平都不同程度的高于未分化的细胞。结论:运用CRISPR-Cas9基因编辑技术能够实现对人胚胎干细胞的特定的编辑;MSX1基因敲除hESc还能保持干细胞的多能性并向三胚层细胞分化。我们获得的MSX1基因敲除hESc可以用于进一步探讨牙齿发育的分子机制,进而为人类研究MSX1基因异常导致的先天缺牙等相关疾病的治疗提供一定参考。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R78
【参考文献】
相关期刊论文 前9条
1 朱姝;盛巍;陈旭;;与先天缺牙相关的全身综合征[J];中国实用口腔科杂志;2016年09期
2 郭晓强;;CRISPR-Cas9技术发展史:25年的科学历程[J];自然杂志;2016年04期
3 陈倩倩;李月;孟郁洁;戚晓鹏;;MSX1基因与非综合征型唇/腭裂关联研究进展[J];中国优生与遗传杂志;2015年12期
4 李聪;曹文广;;CRISPR/Cas9介导的基因编辑技术研究进展[J];生物工程学报;2015年11期
5 刘志国;;CRISPR/Cas9系统介导基因组编辑的研究进展[J];畜牧兽医学报;2014年10期
6 李蓝;张博文;赵志河;;牙缺失基因的研究进展[J];华西口腔医学杂志;2013年04期
7 潘爽;凌均h，
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