Effects Of Insulator SCS 'on Gene Expression Of Baculovirus Gene Transduction Vector In Mammalian Cell Line

Bacterial expression system was newly developed for gene expression due to its special advantages. It owns many unique qualities as a gene expression vector in the mammalian cell. Firstly, the genetic background of type specimen such as ACMNPV is clear and well known. Secondly, due to the disability of reproducing in mammalian cell, it is much safer than other vectors; finally, the vector does little harm to the host cell but could carry considerable exogenous gene.However, baculovirus expression system is not perfect as well currently. There are several problems which need to be solved. For instance, transduction efficiency in many mammalian cell is low; In addition, because baculovirus vector could not reproduce in mammalian cell, the expression is so soon that we may not obtain sufficient protein.We have inserted the SCS’ insulator in our vector to exploit the mechanism of the insulator in mammal cells. At the mean time, we have studied the impact on exogenous gene expression when the SCS’ insulator exists in the vector.SCS’ insulator is one of the earliest discovered insulators. SCS’ means Specialized Chromatin Structure and it is a special chromatin structure found at the distal boundary of the87A7hsp70puff of polytene chromosomes. Bracketing a gene by scs’, it leads to position-independent expression of that gene. And as a result, the gene expression can be enhanced.During the experiment, the SCS’ insulator was obtained through PCR after Drosophila melanogaster DNA extraction. Then we construct nine recombinant baculovirus vectors which contain SCS’ CMV promoter and GFP reporter gene. Also SCS’ insulators in different directions are disposed upstream or downstream from the gene expression cassette CMV-GFP. By analyzing of GFP expression level, GFP expression of one baculovirus vector named VH+is dramatically increased. In VH+, SCS’ insulator is in the same direction as the promoter CMV and setted upstream from the gene expression cassette CMV-GFP. Compared with the control virus, it reaches nearly3-fold24hours after infection in CHO cell lines. While GFP expression level of other recombinant virues decreased in varying degrees. Take one baculovirus vector named VS-H+for example:SCS’insulator upstream from the gene expression cassette CMV-GFP is in the same direction as the promoter CMV and the insulator downstream from the cassette is in the opposite direction as the promoter. Compared with the control virus, GFP expression decreased more than three times. Theoretical study on the mechanisms of the SCS’insulators in mammal cells and the reasons why GFP expression change need to be continued.To sum up, when exploiting the mechanism of insulator in mammal cells, we have selected SCS’insulator to study in baculovirus expression vector system. During the experiment, the exogenous gene expression level was affected obviously by the insulator. The results of the study provide us a new way to optimize the baculovirus vector system. More importantly, It provides a very good material when we further study the mechanism of insulator in mammalian cell lines.