科研之友
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摘要
The pantropic retroviral expression system has been widely used to stably introduce foreign genes into the genomeof dividing cells fromnon-mammalian systems. However, we found that the current commercial retrovirus expression systemdid notwork in inactively dividing primary shrimp cells. To overcomethis, we successfully developed a triple-pseudotyped retroviral expression system that can be used for effective gene transfer and expression of reporter genes in both mammalian and penaeid shrimp cells by improving the commercial pantropic retrovirus system (VPK-305, Cell Biolabs) in two ways. First, the promoter regions of translationally controlled tumor protein (TCTP) genes were cloned from the shrimps Metapenaeus ensis and Marsupenaeus japonicus, named Pme-tctp and Pmj-tctp, respectively. We inserted them into the retroviral reporter vectors of pMCs-P-LTR-GFP or eGFP, immediately after the retroviral promoter P-LTR (5' LTR, long terminal repeat sequence). We then analyzed their expression efficiencies in mammalian and shrimp cells by the lipofection method. It was found that P-LTR was a strong promoter in mammalian cells, but failed to effectively drive the reporter genes to express in the primary lymphoid cells (LCOO) derived from Oka organs of shrimp (M. ensis). However, inclusion of the shrimp promoters (Pme-tctp and Pmj-tctp) significantly improved the expression efficiency of the retroviral vectors in shrimp cells. Next, the revised retroviral vectors of pMCs-P-LTR-Pme-tctp (or Pmj-tctp)-GFP or eGFP were pseudotyped with envelope protein VSV-G alone, or triplywith VSV-G, VP19 and VP28 (two envelope proteins of shrimp white spot syndrome virus). And we then examined the corresponding infection and expression efficiencies in shrimp primary LCOO cells and embryo cells. We found that only the triple-pseudotyped retrovirus containing the shrimp-derived promoter could successfully mediate the delivery and fluorescent expression of GFP and eGFP genes, but VSV-G-pseudotyped retrovirus had poor tropism to shrimp LCOO cells. Confocal microscopic observation further confirmed the hypothesis that the improved tropism of the triplepseudotyped retrovirus to shrimp cells might be attributed to the inclusion of VP19 and VP28 into the envelope of the packaged retrovirus. In spite of this, the expression efficiency and infection efficiency (20%-30%) of reporter genes in shrimp cells was still lower than those in mammalian cells. In infected shrimp primary embryo cells, strong green fluorescent signal was detected only in neuron-like cells, not in fibroblast-like cells. This triplepseudotyped retroviral expression system ultimately may prove useful for immortalization of cultured shrimp cells and production of transgenic shrimps. Statement of relevance: We developed a novel triple-pseudotyped retroviral expression systemthat can be used in effective gene transfer and expression of foreign genes in shrimp cells. This system may prove useful for the immortalization of cultured shrimp cells which are needed in the study and control of shrimp viral diseases. This system may prove useful for the production of transgenic shrimps with new traits.
- 出版日期2017-1-20
- 单位中国海洋大学
