Application Of The Transgenic Vectors With Dual Fluorescent Reporter Genes

Since being discovered, green fluorescent protein and red fluorescent protein were used widely in many biological research fields because of their easy detection, fluorescence stablity, non-toxic, species universal, easy construction, real-time location in living cells and so on. Their applications involved in every field of life science, including the research of regulatory elements, enhaner capture, protein localization of gene expression, expression phase, distribution, molecular mechanisms of axiology, screening drugs, clinical testing, labeling of the transgenic animals and plants, localization of virosome, interaction of proteins and so on. Therefore we constructed different transgenic vectors with dual fluorescent reporter genes for different purposes.First, in order to explore the activy of the bluntnose black bream (Megalobrama amblycephala)β-actin promoter (PAβ)in silkworm (Bombyx mori) BmN cells and in vivo, we transferred the transgenic vector pigA3GFPAβDsRed and helper plamid into the BmN cells by the liposome-mediated gene transfer method and into the silkworm eggs by sperm-mediated gene transfer method respectively. 48 hours later, both green fluorescence and red fluorescence were observed in the BmN cells and 7 days later the two kinds of fluorescence were detected in the fertilized eggs of the silkworm. These results suggested that PAβof bluntnose black bream could work in BmN cells. With the steady passage of the transformed cells, the green fluorescence existed stably but the red fluorescence reduced gradually, suggested that the activity of PAβin the BmN cells was weakened because of its susceptible to some cytoline in the BmN cells.Second, to explore the applicability of the transposon piggyBac derived insects, two transgenic vectors pigA3GFPAβDsRed and pigA3GFP-CMVDsRed-IEneo based on transposon piggyBac were constructed. Both of the green fluorescence and the red fluorescence were detected in the transfected Ctenpharyngoden Idellus kidney (CIK) cells with the transgenic vectors and the helper vector pigA3 or pigCMV in which the transpoase gene was driven by cytomegalovirus (CMV) promoter (PCMV). Moreover the transgenic vector pigA3GFP and pigA3GFPAβDsRed were introduced into eggs of the goldfish (Carassius auratus) by sperm-mediated gene transfer. Finally the transgenic pigA3GFP and pigA3GFPAβDsRed goldfishes were obtained and verified by PCR and Southern blotting, among which the one 2-month-old transgenic pigA3GFPAβDsRed goldfish was dissected and both the green fluorescence and the red fluorescence could be detected in several organs, therefore we suggested that the transposon piggyBac was a potentially powerful tool for producing transgenic fish.Third, to explore the profile of immobilization of Bombyx mori nuclear polyhydrosis virus (BmNPV) polyhedron, we firstly constructed both gfp and DsRed contained vector pIZT-DsRed which was used to transfect BmN cells to generate a stable pIZT-DsRed-transformed BmN cell line by screened with zeocin. Then the wild type BmNPV was employed to infect the cell line most of which expressed gfp and DsRed simultaneously so as to observe newly formed polyhedron. The results of emitting green fluorescence and red fluorescence from isolated BmN polyhedron revealed that foreign proteins could be embedded into the polyhedron on a nonspecific recognition way.