果蝇SCS'绝缘子对杆状病毒在哺乳动物细胞中介导基因表达的影响

The Effect of Drosophila SCS' Insulator on Transgene Expression Mediated by Baculovirus in Mammalian Cells

将果蝇SCS'绝缘子以不同方向置于CMV启动子和报告基因egfp表达框的上下游,构建了包括对照病毒在内的9个重组杆状病毒.比较了这些病毒转导CHO,BHK和CNE细胞后报告基因表达的情况.结果显示:各株重组病毒的复制没有明显差异,但报告基因的表达水平有明显不同.当SCS'绝缘子位于表达框的上游,并与报告基因同向时,基因的表达水平显著提高.在CHO中,转导后24hEGFP表达量比对照病毒提高近3倍,egfp mRNA水平也比对照病毒高4倍多.当在表达框上、下游各有一个SCS',且分别与CMV启动子方向一致和相反时,EGFP表达量降低到只有对照病毒的1/3,其mRNA水平也仅是对照病毒的1/6～1/5.丁酸钠和TSA等组蛋白脱乙酰基酶抑制剂处理可以使该病毒介导EGFP表达大幅上升,甚至超过对照病毒.这些结果揭示了杆状病毒在哺乳动物细胞介导基因表达时受到细胞染色质调节机制的影响,也为优化杆状病毒哺乳动物细胞系基因表达系统提供了新的方法.

Drosophila SCS＇ insulator was inserted upstream, downstream, or both up-and down-stream of an egfp gene expression cassette driven by CMV promoter, in different directions. They were then inserted into baculovirus genome to construct eight recombinant viruses, plus a control virus. The expression of egfp was compared in mammalian cell lines CHO, BHK and CNE transduced by these viruses. Although these viruses replicated to a similar level in insect Sf9 cells, the level of egfp expression mediated by these viruses varied significantly in virus-transduced mammalian cells. The ,expression level was highest when SCS＇ was placed upstream of the expression cassette in the same orientation as egfp gene. In CHO cells, EGFP level expressed by this virus was almost 3 times of the control virus, while the egfp mRNA was also 4 times higher than the control virus. When one SCS＇ was inserted upstream of the egfp gene in the same direction as the gene, while another SCS＇ downstream of the egfp gene in the Opposite direction, the expression level of EGFP was only 1/3 of the control virus, and the egfp mRNA as 1/6-1/5 of the control virus too. Presence of Sodium butyrate and TSA, as the inhibitors of histone deacetylase in the medium, singnicantly improved the expression of EGFP by this low expression virus, with the expression level surpassed the control virus. These results indicated that the gene expression mediated by baculovirus was affected by the regulating mechanism of cellular chromatin structure, and provided an approach to improve the expression effect of baculovirus as a mammalian cell gene expression vector.