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Researches on the Construction of EphA3 Gene Stable-expressed Cell Models

Author: LiQiMan
Tutor: PanDaRen；ZhouJianGuang
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: EphA3 gene PC-1 gene NIH3T3 LNCaP stable cell lines functional analysis GST-fusion plasmid
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 14
Quote: 0
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Abstract


EphA3 is a new gene, and the reference about EphA3 is scarcely. The previous research in our laboratory found that the expression of EphA3 was relevant to that of PC-1 gene. This relevance stimulates us to think that EphA3 may play some role in the progress of prostate cancer.Therefore, eukaryotic expression plasmid, that is, pcDNA3.1(-)/myc-his-EphA3, was constructed in order to examine our hypothesis. Then NIH3T3-EphA3、NIH3T3-null cell lines were acquired through stable cell transfection. Finally the MTT, plate colony formation assay, soft agar colony formation were used respectively to observe the effect of EphA3 gene expression on tumor development and progression in NIH3T3. The results show that extraneous EphA3 gene stably expressed mouse fibroblast cell line was successfully established. The integration and expression of extraneous EphA3 gene in NIH3T3 was confirmed by western blot analysis. Comparing with the parental cells and the cells tranfected with empty vector, the EphA3 gene transfectants acquired the ability of forming clone quickly and growing on soft agar.Moreover, pcDNA3.1-EphA3-IRES-PC-1 was also constructed for the comprehensive understand of EphA3 gene through comparing with PC-1 gene. As a result, EphA3 overexpression cell line LNCaP-EphA3, PC-1 overexpression cell line LNCaP-PC-1, EphA3 and PC-1 overexpression simultaneously cell line LNCaP-EphA3-PC-1 were established at the same time. Then observed the effect of EphA3/ PC-1 through the methods as above.The result demonstrated that EphA3 can play an important role in the progression of prostate cancer, which acted like PC-1. All the results revealed that EphA3 gene is a positive-relevance gene of prostate cancer, and that it can stimulate the prostate cancer move to more serious levels.In addition, the serial functional assay showed that EphA3 gene was oncogene, but the molecular mechanisms of such function were also kept in a closed box. Thus nine plasmids were constructed in order to reveal the course of signal pathways. And four of them were induced by IPTG successfully, which will be used for the assay of GST- Pull down, CO-IP.

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