本文選題：Dlk1-Dio3印記區(qū)域 + 差異甲基化區(qū)域��； 參考：《哈爾濱工業(yè)大學》2016年碩士論文

【摘要】：Dlk1-Dio3印記基因簇位于小鼠的第十二號染色體末端(12qF1),以及人類的第14號染色體上(14q32)。該印記區(qū)域對胚胎和胎盤的生長發(fā)育起到重要的調控作用,還影響成體的新陳代謝和大腦的功能。且該區(qū)域內各基因表達在生長發(fā)育中呈現一個動態(tài)變化的過程。CGI-2位于該區(qū)域內miR-341和miR-1188附近,為該區(qū)域內首個被發(fā)現的母本甲基化而父本非甲基化的差異甲基化區(qū)域,且為結合CTCF并具有絕緣子活性的DNA功能原件。因此,本文選取Dlk1-Dio3印記區(qū)域為研究對象,采用CRISPR/Cas9基因編輯技術對CGI-2進行長片段的基因組刪除,在細胞水平上分析CGI-2敲除后其上下游基因的表達變化。首先,本文對細胞系內Dlk1-Dio3印記區(qū)域中各基因表達水平進行分析,從而篩選出三株適合敲除實驗的細胞系。所選擇的三株細胞系分別為Dlk1-Dio3印記區(qū)域中各基因表達水平偏高的N2a、該印記區(qū)域中各基因表達水平較低的Hepa1-6以及非癌細胞系NIH3T3。通過向導RNA預測網站設計出用于CRISPR/Cas9敲除實驗的兩對向導RNA,并且構建px330重組質粒。其次,在N2a細胞中進行CGI-2敲除預實驗,選擇適合用于實驗的向導RNA。隨后,分別在N2a、Hepa1-6和NIH3T3三種細胞系中進行CGI-2的CRISPR/Cas9的基因敲除,在驗證敲除成功的前提下通過實時定量分析的方法判斷在CGI-2缺失的情況下Dlk1-Dio3印記區(qū)域內父母本基因的表達水平變化。其結果顯示,敲除位點遠端的Dio3并沒有明顯的變化、Dlk1有不顯著的下調趨勢;位于次遠端的Mirg呈現出上調的趨勢,但在不同細胞系中其上調程度存在差異;而位于區(qū)域中間部分的Gtl2、Rtl1、Rian、Meg8、Irm和AB063319有顯著的上調趨勢。綜上可知,CGI-2的缺失會導致Dlk1-Dio3印記區(qū)域中各基因表達水平的變化,即CGI-2對該印記區(qū)域的表達具有調控作用。最后,檢驗在刪除CGI-2和未刪除CGI-2的兩種情況下Dlk1-Dio3印記區(qū)域內Dlk1-DMR、Gtl2-DMR、IG-DMR和CGI-1-DMR這四個差異甲基化區(qū)域的甲基化狀態(tài)。結果顯示,在刪除CGI-2和未刪除CGI-2的兩種情況這四個差異甲基化區(qū)域均為高甲基化狀態(tài),即未發(fā)生改變。綜上所述,CGI-2的缺失會導致其所在的Dlk1-Dio3印記區(qū)域內多種基因表達水平發(fā)生變化,且這種變化趨勢在不同細胞系中大致相同;CGI-2對該印記區(qū)域中基因表達的調控作用與該區(qū)域內其他差異甲基化位點的甲基化狀態(tài)無關。
[Abstract]:The Dlk1-Dio3 imprinting gene cluster is located at the end of chromosome 12 in mice and on chromosome 14 in humans.The imprinted region plays an important role in regulating the growth and development of the embryo and placenta, and also affects the metabolism of adults and the function of the brain.The expression of genes in the region showed a dynamic process during growth and development. CGI-2 was located near miR-341 and miR-1188 in the region, which was the first differential methylation region in which the female parent was found to be methylated and the male parent was demethylated.And it is the original of DNA function which binds CTCF and has insulator activity.Therefore, the Dlk1-Dio3 imprinting region was selected as the research object, CRISPR/Cas9 gene editing technique was used to delete the long fragment of CGI-2 genome, and the expression changes of upstream and downstream genes after CGI-2 knockout were analyzed at the cellular level.Firstly, the expression levels of genes in the Dlk1-Dio3 imprinting region of the cell line were analyzed, and three cell lines suitable for knockout test were selected.The three cell lines selected were N2a with higher gene expression level in the Dlk1-Dio3 imprinting region, Hepa1-6 with lower gene expression level in the imprinted region and NIH3T3, a non-cancer cell line.Two pairs of wizards RNAs for CRISPR/Cas9 knockout experiment were designed by guided RNA prediction site, and px330 recombinant plasmid was constructed.Secondly, the CGI-2 knockout pretest was carried out in N2a cells, and the suitable guide for the experiment was selected.Then, the gene knockout of CGI-2 CRISPR/Cas9 was performed in N2aHep Hepa1-6 and NIH3T3 cell lines, and the expression level of parental gene in Dlk1-Dio3 imprinted region was determined by real-time quantitative analysis on the premise of verifying the success of knockout.The results showed that there was no significant change in Dio3 of the distal knockout site, and the down-regulation of Dlk1 was not significant. The Mirg located at the subdistal end showed an up-regulation trend, but there were differences in the degree of upregulation in different cell lines.In the middle part of the region, Gtl2Rtl1, Rtl1, Rtl1, Meg8, Irm and AB063319 showed a significant upward trend.It can be concluded that the absence of CGI-2 will lead to the change of gene expression level in the Dlk1-Dio3 imprinting region, that is, CGI-2 can regulate the expression of the imprinted region.Finally, the methylation status of the four differential methylation regions of the Dlk1-Dio3 imprinted region (Dlk1-DMR-Gtl2-DMR-IG-DMR and CGI-1-DMR) was examined under the condition of deleting CGI-2 and not deleting CGI-2.The results show that the four differential methylation regions are hypermethylated in the case of deleting CGI-2 and not deleting CGI-2, that is, no change has taken place.In conclusion, the absence of CGI-2 may result in changes in the expression levels of multiple genes in the Dlk1-Dio3 imprinting region.The effect of CGI-2 on gene expression in the imprinted region was not related to the methylation status of other differentially methylated sites in different cell lines.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78
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本文編號：1758605