The frequent outbreak of viral diseases in the intensively cultured shrimps hasmarkedly limited the development of shrimp aquaculture in the world. Shrimp cellline is a good tool to separate and purify the shrimp virus, to study the pathogenicmechanism of the virus and to develop the effective diagnostic reagents. Despitenumerous attempts, no continuous shrimp cell line has yet been established.Therefore,it is necessary to improve the present techniques of primary culture,subculture and gene transfer of shrimp cells. This will lay a solid foundation for theworks on immortalization transformation of shrimp cells as well as the establishmentof continuous shrimp cell line.Firstly, based on the previously published data on the media used for shrimpcell culture, three new shrimp media were formulated:0.2×L-15based shrimpmedium I,2×L-15based shrimp medium Ⅱ and1.5×L-15based shrimp medium Ⅲ,and compared their capability to support the growth and survival of shrimp lymphoidcells. Of the three media tested, the1.5×L-15-based shrimp medium Ⅲ (containd15%fetal bovine serum,10%Shrimp muscle extraction,10μg·mL-1amino acid,20μg·mL-1growth factor,100IU·mL-1Penicillin G sodium and100μg·mL-1Streptomycin) gave the best results. The cell migration from the tissue explants wasinitiated most quickly in the shrimp medium Ⅲ (at2~3h after seeding). Shrimp cellmonolayer of70%~80%confluence could be formed within16~24hours in theshrimp medium Ⅲ. And also the best survival of shrimp cells was achieved inshrimp medium Ⅲ for28±3days. Moreover, the reseeded lymphoid explants couldre-attach into the well and produced a new outgrowth around them due to the active migration of cells in shrimp medium Ⅲ. In addition, we first developed a reliabledisinfection protocol for the preparation of sterile lymphoid organs.Meanwhile, the susceptibility of the primary lymphoid cell cultures to WSSVwas also detected. Within two days post infection, apparent cytopathic effect (CPE)was observed in the primary shrimp cell cultures inoculated by higher WSSV titer of4×10~7,4×10~6and4×10~5virus per μl. The infected cells initially exhibited shrinkageor became aggregated. And5days later, most of the infected cells rounded up andthen detached from the culture dishes or broke up. In contrast, no obvious CPE wasobserved in the lymphoid cell cultures which were inoculated with the lower titer ofWSSV dilutions, same volume of PBS or heat-inactivated WSSV. The susceptibilityof the primary lymphoid cell cultures to WSSV also indicated that the newlyformulated shrimp medium Ⅲ was suitable for the growth and survival of the shrimpcells.Secondly, thirteen methods including eight digestive enzymes or reagents, twophysical methods of4℃PBS shock and cell scraper, and three combined methodsof enzyme-reagent and enzyme/reagent-4℃PBS shock were tried to subculture theprimary shrimp cell cultures in this study. And the subculture efficiencies includingthe time and percentage needed for both detachment and reattachment of primaryshrimp cell cultures by these methods were recorded and compared. It was foundthat, the non-mammalian-derived enzyme complex of HyQTase and the enzyme-freecell dissociation solution (ECDS) showed higher subculture efficiency than the otherenzymes and physical methods did. For HyQTase, about80%~89%of the primarycell monolayer could be detached by the stock solution in6minutes and49.5±1.5%of the dislodged cells could reattach to the well substrate. In contrast, dilutions of50%and25%stock solutions of HyQTase were less effective with longerdetachment time of8and10mins, lower detachment efficiency of60%~69%(forboth dilutions) and plating efficiency of31.7±3.8and25.6±4.0, respectively. Similarresults were obtained in the use of ECDS solution, that is, stock solution was moreeffective than the two dilutions, and produced a similar plating efficiency (50.3±2.7%) with HyQTase. And when HyQTase and ECDS solution werecombined with the4℃PBS shock separately, their subculture efficiency could bemarkedly increased with shorter detachment time of4.5mins, higher detachmentefficiency of90%~100%(for both), and higher plating efficiency of54.1±2.0%forHyQTase and60.2±4.9%for ECDS. Using the above-mentioned methods, we couldsubculture the lymphoid cell cultures twice. The widely-used0.25%trypsin couldeasily detach the shrimp cells, but the dislodged cells had some trouble to reattachand survive. However,0.125%trypsin gave much higher reattachment efficiency(45.7±1.0%) under the same detachment efficiency of80%~90%, and thus produceda subculture efficiency immediately after ECDS and HyQTase did. In contrast, thedose of0.05%trypsin was too low to successfully passage the shrimp cell monolayer.In addition to trypsin, the other five digestive enzymes of collagenase Ⅱ, dispase,hyaluronidase, elastase and pronase also produced heavy damages to the shrimp cellsand resulted in bad subculture effects at all the three doses tested. Of the fivedigestive enzymes, collagenase Ⅱ gave the best subculture effects. In contrast to2mg·ml-1collagenase Ⅱ, the lower dose of1mg·ml-1collagenase Ⅱ was more suitablefor the subculture of shrimp cells due to its significant higher plating efficiency of36.2±7.3%. Noteworthy, the other four digestive enzymes were quite unsuitable forthe subculture of shrimp cells due to the relatively long detachment time of19~30mins and quite low plating efficiency of0.9%~21%. The physical methods of4℃PBS shock or use of cell scraper alone were found to be less effective in thesubculture of shrimp cells for their relatively low plating efficiency of25.0±10.0%and24.7±9.0%, respectively.Finally, the gene transfer techniques for the primary lymphoid cells from thegreasyback shrimp Metapenaeus ensis were explored. Liposome transfection andretrovirus-mediated gene transfer systems are commonly used methods to transferthe exogenous genes into the cells. C-Myc gene is one of the four transcriptionfactors used for molecular reprogramming to induce adult cells into iPS cells, and itplays a key role in regulating the growth, proliferation and immortal transformation of cells. In this study, the two above-mentioned methods were used to transfer themouse c-Myc gene into lymphoid cells of the greasyback shrimp. The total genomeDNA and total RNA were extracted from the shrimp lymphoid cells at the4th dayafter transfected by the expression plasmid pMXs-c-Myc or infected by retrovirusvirion containing vector pMXs-c-Myc. And a pair of c-Myc-specific primers wasused to amplify the c-Myc gene to confirm its integration and expression. The resultsobtained showed that, the c-Myc gene can be introduced into the primary shrimplymphoid cells by liposome transfection method, but the introduced c-myc geneswere not transcripted and expressed in the shrimp cells. The failure of the CMV(Cytomegalovirus) promoter of pMXs-c-Myc plasmid to be activated in shrimp cellsmay result in the silence of integrated c-Myc in shrimp cells. Retrovirus-mediatedgene transfer method failed to introduce the c-Myc gene into the shrimp lymphoidcells, and also no transcription and expression of c-Myc was detected. The lack ofmammalian retrovirus receptors in the cellular membrane of the shrimp lymphoidcells maybe the reason why the retrovirus cannot mediate the transfer of c-Myc geneinto the genome of the shrimp cells.In order to verify the above-mentioned results, a GFP reporter plasmid ofpMCs-GFP was also introduced into the shrimp lymphoid cells by the methods ofliposome transfection and retrovirus-mediated gene transfer system. Under thefluorescence microscope, the expression of GFP was observed. It was found that thecontrol explants of shrimp lymphoid organs without GFP transfection producedobvious green fluorescence from the first day of seeding. In contrast, the shrimplymphoid cell monolayer did not produce green fluorescence within the first4days,but weak green fluorescence appeared from the fifth day after seeding. Therefore,the detection of the expression of the GFP gene should be carried out within the first4days after seeding, otherwise, the spontaneous background fluorescence willinterrupt the results, and the background fluorescence should be taken intoconsideration. In the GFP-transformed shrimp lymphoid cells, GFP expression wasnot observed no matter liposome transfection or retrovirus (GFP) infection was used. This result is consistent with the result of c-Myc gene transformation.In conclusion, the formulated1.5×L-15based shrimp medium Ⅲ gave the bestresults in the lymphoid cells migration from the tissue explants, the time of shrimpcells monolayer of70%~80%confluence, the survival time and the state of the cells.HyQTase and ECDS solution combined with the4℃PBS shock separately are theeffective methods to subculture the primary cells from the lymphoid organs ofgreasyback shrimp. C-Myc gene can be introduced into the primary shrimplymphoid cells by liposome transfection method, but the introduced c-myc genescould not be transcribed and expressed. Retrovirus-mediated gene transfer methodfailed to introduce the c-Myc gene into the shrimp lymphoid cells. And the aboveresults are confirmed by GFP gene transformation. The above-mentioned data maylay a solid foundation for the further works on the immortalization of shrimp cellsand the development of continuous shrimp cell line. |