锁式探针滚环扩增方法在小鼠F9单细胞基因表达研究中的应用
发布时间:2019-01-06 06:32
【摘要】:基因表达检测与分析是研究细胞功能的重要手段之一,但传统的分析方法通常是以细胞群体为研究对象,得到的是细胞群体mRNA的整体或平均表达水平,缺少针对单细胞中mRNA定位和定量分析的有效手段。锁式探针滚环扩增(Padlock rolling-circle amplification, Padlock-RCA)原位检测技术是基于锁式探针与单链DNA靶点的特异性结合,连接转换为环形分子后滚环扩增原位检测单链DNA靶点。该技术具有高特异性和高敏感性,能定位和定量分析单细胞内DNA靶点,因此与PCR和传统原位杂交技术相比有明显优势。Padlock-RCA是一系列反应的整合,首先,利用特异的锁核酸(Locked nucleic acid, LNA)与mRNA结合,可在细胞内原位将mRNA反转录成cDNA,通过RNase H将mRNA降解的同时,锁式探针(Padlock probe)与新合成的cDNA中靶序列结合,并通过连接酶的作用,锁式探针自连环化,形成单链环化模板。随后在Phi29聚合酶的作用下,对环化产物进行滚环扩增(RCA),最后荧光探针与扩增产物上的靶序列特异性结合,从而实现对细胞内mRNA的表达水平的检测。Padlock-RCA对组织细胞中含量极低的mRNA有极高的敏感性,能够在单细胞水平检测mRNA的表达,因此可以实现在细胞群体中发现某些基因特异表达的罕见细胞和细胞亚群。 本研究的目的就是优化并利用锁式探针滚环扩增方法对小鼠畸胎瘤细胞F9单个细胞中的基因表达进行原位检测。研究主要包括四个部分,首先建立滚环复制扩增的方法,通过对随机引物,Phi29聚合酶,扩增时间等方面的条件摸索,建立了稳定、高效的滚环复制扩增体系;其次,优化锁式探针滚环扩增方法并检测小鼠畸胎瘤细胞F9单细胞中Nanog基因及Perl基因的表达;而后利用维甲酸(RA)诱导小鼠畸胎瘤细胞F9分化模型,对细胞分化前后Nanog基因mRNA表达水平的改变进行分析;最后将F9细胞通过皮下注射,接种到129SV小鼠体内,待成瘤后,将畸胎瘤组织制备冰冻切片,利用Padlock-RCA原位检测小鼠畸胎瘤组织中Nanog基因的表达。本研究成功的应用锁式探针滚环扩增方法检测了Nanog基因和Perl基因在部分F9单细胞中的表达;并发现Nanog基因在F9细胞分化前后表达水平的差异;同时在小鼠畸胎瘤冰冻组织切片中发现大量Nanog基因表达阳性的细胞。为进一步研究单细胞基因表达及细胞亚群功能分析开辟了一条新途径。
[Abstract]:The detection and analysis of gene expression is one of the important methods to study cell function, but the traditional analysis method usually takes the cell population as the research object, and obtains the whole or average expression level of mRNA in the cell population. Lack of effective means for localization and quantitative analysis of mRNA in single cell. The technique of lock-type probe rolling amplification (Padlock rolling-circle amplification, Padlock-RCA) in situ detection is based on the specific binding of the lock-type probe to the single-stranded DNA target, and the ligation is transformed into the ring molecular roller-loop amplification in situ detection of single-stranded DNA target. This technique has high specificity and sensitivity, and can locate and analyze the target of DNA in single cell, so it has obvious advantages over PCR and traditional in situ hybridization. Padlock-RCA is the integration of a series of reactions. By using specific lock-nucleic acid (Locked nucleic acid, LNA) binding to mRNA, mRNA could be inverted into cDNA, in situ to degrade mRNA by RNase H, and the lock-probe (Padlock probe) could bind to the target sequence of the newly synthesized cDNA. The single chain cyclization template was formed by self-linking of the lock-type probe by ligase. Then under the action of Phi29 polymerase, the cyclized product was rolled to amplify the (RCA), and the final fluorescent probe was specifically bound to the target sequence of the amplified product. Padlock-RCA is highly sensitive to mRNA, which is very low in tissue cells, and can detect the expression of mRNA at single cell level. Therefore, rare cell and cell subsets with specific expression of some genes can be found in cell populations. The aim of this study was to optimize and detect in situ gene expression in single mouse teratoma cell line F9 by locking probe rolling amplification method. The research mainly includes four parts. Firstly, the method of rolling loop replication and amplification was established. By exploring the conditions of random primer, Phi29 polymerase, amplification time and so on, a stable and efficient rolling loop replication amplification system was established. Secondly, the Nanog gene and Perl gene expression in mouse teratoma cell F9 single cell were detected by optimizing the method of loop amplification with locking probe. Then the F9 differentiation model of mouse teratoma cells induced by retinoic acid (RA) was used to analyze the changes of mRNA expression of Nanog gene before and after differentiation. Finally, F9 cells were injected subcutaneously into 129SV mice. After tumorigenesis, frozen sections of teratoma tissue were prepared and the expression of Nanog gene in mouse teratoma tissue was detected by Padlock-RCA in situ. In this study, the expression of Nanog gene and Perl gene in F9 single cell was detected by the method of locking probe rolling amplification, and the difference of Nanog gene expression before and after F9 cell differentiation was found. At the same time, a large number of Nanog gene positive cells were found in frozen tissue sections of mouse teratoma. It opens a new way for further study of single cell gene expression and functional analysis of cell subsets.
【学位授予单位】:北京协和医学院
【学位级别】:硕士
【学位授予年份】:2011
【分类号】:R346
本文编号:2402468
[Abstract]:The detection and analysis of gene expression is one of the important methods to study cell function, but the traditional analysis method usually takes the cell population as the research object, and obtains the whole or average expression level of mRNA in the cell population. Lack of effective means for localization and quantitative analysis of mRNA in single cell. The technique of lock-type probe rolling amplification (Padlock rolling-circle amplification, Padlock-RCA) in situ detection is based on the specific binding of the lock-type probe to the single-stranded DNA target, and the ligation is transformed into the ring molecular roller-loop amplification in situ detection of single-stranded DNA target. This technique has high specificity and sensitivity, and can locate and analyze the target of DNA in single cell, so it has obvious advantages over PCR and traditional in situ hybridization. Padlock-RCA is the integration of a series of reactions. By using specific lock-nucleic acid (Locked nucleic acid, LNA) binding to mRNA, mRNA could be inverted into cDNA, in situ to degrade mRNA by RNase H, and the lock-probe (Padlock probe) could bind to the target sequence of the newly synthesized cDNA. The single chain cyclization template was formed by self-linking of the lock-type probe by ligase. Then under the action of Phi29 polymerase, the cyclized product was rolled to amplify the (RCA), and the final fluorescent probe was specifically bound to the target sequence of the amplified product. Padlock-RCA is highly sensitive to mRNA, which is very low in tissue cells, and can detect the expression of mRNA at single cell level. Therefore, rare cell and cell subsets with specific expression of some genes can be found in cell populations. The aim of this study was to optimize and detect in situ gene expression in single mouse teratoma cell line F9 by locking probe rolling amplification method. The research mainly includes four parts. Firstly, the method of rolling loop replication and amplification was established. By exploring the conditions of random primer, Phi29 polymerase, amplification time and so on, a stable and efficient rolling loop replication amplification system was established. Secondly, the Nanog gene and Perl gene expression in mouse teratoma cell F9 single cell were detected by optimizing the method of loop amplification with locking probe. Then the F9 differentiation model of mouse teratoma cells induced by retinoic acid (RA) was used to analyze the changes of mRNA expression of Nanog gene before and after differentiation. Finally, F9 cells were injected subcutaneously into 129SV mice. After tumorigenesis, frozen sections of teratoma tissue were prepared and the expression of Nanog gene in mouse teratoma tissue was detected by Padlock-RCA in situ. In this study, the expression of Nanog gene and Perl gene in F9 single cell was detected by the method of locking probe rolling amplification, and the difference of Nanog gene expression before and after F9 cell differentiation was found. At the same time, a large number of Nanog gene positive cells were found in frozen tissue sections of mouse teratoma. It opens a new way for further study of single cell gene expression and functional analysis of cell subsets.
【学位授予单位】:北京协和医学院
【学位级别】:硕士
【学位授予年份】:2011
【分类号】:R346
【参考文献】
相关期刊论文 前3条
1 ;Isolation and characterization of the murine Nanog gene promoter[J];Cell Research;2005年05期
2 ;Roles of the Nanog protein in murine F9 embryonal carcinoma cells and their endoderm-differentiated counterparts[J];Cell Research;2006年07期
3 朱惠芳,朱苏玲,蒋耀青;维生素A酸诱导小鼠胚胎性癌细胞分化过程中核仁形成区的变化[J];遗传;1990年06期
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