title: Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines.
abstract: We have constructed a series of plasmid vectors for the expression of foreign genes in insects or insect cell lines. We incorporated the Drosophila hsp70 and actin 5C promoters, as well as the hr5 enhancer-driven baculovirus ie1 promoter, into plasmids that allow convenient cloning of heterologous genes into multiple cloning sites. We combined these promoters with either a short, double poly-adenylation site derived from the Heliothis virescens p63 chaperonin gene, or with a fusion of the small t intron with the early 3' untranslated region and poly-adenylation sites of SV40. Unique eight base cutter restriction sites flanking the promoters and poly-adenylation sequences make it possible to transfer the entire transcription units into other sequence contexts, for example, into transposable elements or into other plasmids bearing selectable marker genes. It is also convenient to combine two of our transcription units on the same plasmid in order to express multiple genes simultaneously. To test the ability of our vectors to drive expression of reporter genes, luciferase derivatives were made of the expression plasmids and introduced into Aedes albopictus C6/36 cells by electroporation or into Anopheles gambiae embryos by biolistic particle bombardment. All three promoters directed high levels of luciferase expression. However, there were differences in their relative activities in the two experimental systems. In C6/36 cells, the actin 5C and hr5-ie1 promoters were significantly more active than the hsp70 promoter. In Anopheles embryos, hsp70 and actin 5C had maximal activities, while hr5-ie1 was weaker. We also found that the constructs containing the SV40 small t intron and early 3' untranslated region sequences had higher expression levels than their counterparts containing the Heliothis poly-adenylation sequence. Our most active construct combines the actin 5C promoter with the SV40 intron and 3' untranslated region sequences. This vector was also used to drive expression of a visible marker, the enhanced green fluorescent protein gene, resulting in readily visible green fluorescent protein expression in C6/36 cells.
mesh_terms: D020413:3' Untranslated Regions; D000199:Actins / Q000235:genetics; D000330:Aedes / Q000166:cytology; D000818:Animals; D030801:Animals, Genetically Modified; D000852:Anopheles / Q000166:cytology / Q000196:embryology; D019470:Biolistics; D002460:Cell Line; D018833:Chaperonins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004331:Drosophila melanogaster / Q000235:genetics; D018274:Electroporation; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D007313:Insecta / Q000166:cytology* / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009036:Moths / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic; D013539:Simian virus 40 / Q000235:genetics