Chicken HS4 insulator significantly improves baculovirus-mediated foreign gene expression in insect cells by modifying the structure of neighbouring chromatin in virus minichromosome.
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is widely used as a eukaryotic expression vector for protein production. In the current study, chicken beta-globin 5'-HS4 insulator (HS4) was placed downstream of the polyhedrin promoter-directed foreign gene expression cassette in AcMNPV, and found to markedly increase the expression of target gene. When enhanced green fluorescence protein gene (egfp) was used as the reporter gene, cells infected by the recombinant virus with HS4 (AcEGFP-HS4) showed 3.0 and 2.1-fold stronger fluorescence than that by the control virus without HS4 (AcEGFP) at 72 and 96 h post infection, respectively. The level of egfp mRNA was also much higher in cells infected by AcEGFP-HS4 than that by AcEGFP. An increase in gene expression was also seen when firefly luciferase gene or secreted alkaline phosphatase gene was used as a reporter. The insertion of HS4 in the polyhedrin locus has no significant effect on virus replication. The effect of HS4 was orientation-dependent, and sensitive to inhibitors of histone acetyltransferase. In DNase I sensitivity assay, HS4 significantly increased the sensitivity of neighbouring DNA to nuclease, but had little effect on DNA of a distal locus. These results suggested that HS4 insulator might affect baculovirus gene expression by modifying the structure of neighbouring chromatin in the virus minichromosome.
Wang
Xin
X
Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, PR China.
Li
Li
L
Ding
Siyuan
S
Huang
Xiaoxia
X
Zhang
Jing
J
Yin
Juan
J
Zhong
Jiang
J
D000107:Acetylation; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D002645:Chickens; D002843:Chromatin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D003474:Curcumin / Q000494:pharmacology; D003850:Deoxyribonuclease I / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D038101:Insulator Elements; D008856:Microscopy, Fluorescence; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D013729:Terpenes / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000235:genetics; D055544:beta-Globins / Q000096:biosynthesis / Q000235:genetics*
D002843:Chromatin; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013729:Terpenes; D014759:Viral Envelope Proteins; D055544:beta-Globins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003850:Deoxyribonuclease I; D003474:Curcumin; C054597:garcinol