Browsing by Subject "Recombinant Baculovirus"

In recent years, animals have been recognized as promising next-generation protein production systems. Animal transgenesis has been achieved primarily in insect cells infected by recombinant baculoviruses. Baculovirus Expression Vector Systems (BEVS) transform the DH10Bac strain of Escherichia coli with the shuttle vector to produce recombinant baculovirus carrying the cargo of interest. The cargo includes at least one promoter driving the expression of at least one protein. PGK is a strong promoter that is naturally active in almost all species where it has been tested, including invertebrates like Drosophila. The PiggyBac transposon-based system is a known strategy for genome integration of foreign genes to create transgenic animals. Nevertheless, nobody has used baculoviruses to deliver genes and produce proteins in earthworms nor to create transgenic earthworms. There is also no information on the sequence of any endogenous E. fetida (earthworm) promoter yet. This project aimed to pilot a novel gene delivery method by creating baculoviruses through the BEVS, carrying the PGK promoter and the GFP reporter gene, and to assess the promoter activity in both Sf9 insect cells and E. fetida through evaluation of GFP fluorescence. Another target was to test the fluorescence after the addition to the baculovirus of the PiggyBac-based inverted terminal repeats (ITRs), flanking the PGK-GFP transcriptional unit. The secondary objective was to develop a non-lethal method for live worm imaging. Conventional restriction enzyme cloning was used to create the shuttle vectors, and restriction digest and Sanger Sequencing were used to identify the positive clones. The Bac-to-Bac BEVS was followed to create baculovirus particles carrying the cargo (PGK-GFP and PGK-GFP-ITR), infect Sf9 insect cells and monitor the PGK activity. Prior to in vitro transfection, the bacmid DNA was confirmed by PCR. These baculoviruses were also used to infect E. fetida and monitor the PGK activity in vivo. E. fetida autofluorescence was assessed before infection. PGK resulted in being much weaker in Sf9 than expected. The flanking of the transcriptional unit of GFP with the ITRs improved the GFP expression. 16% ethanol was shown to anaesthetize E. fetida for 10 to 15 minutes safely. Wild-type and starved E. fetida were shown to have very mild autofluorescence in their digestive system and setae. The coelomic fluid was shown to have strong autofluorescence. Thus, its excretion is crucial before imaging GFP. Likely, all the in vivo fluorescence after infection was due to the worm’s autofluorescence. Therefore, PGK and GFP were unlucky choices for E. fetida.