| Five optimizing experiments include DNA content,liposome content,serum content, cell density and transfection time have been carried out in BmN-SWU1,BmN-SWU2, BmN and Sf21 cell lines by using the cation Lipofectamine 2000 and the expression vector pBac[A3Neo+A3EGFP]. Meanwhile, transgenic cells with high transfecting efficiency were filtered by G418.The results were as follows:1. Optimization of DNA content. We inoculated 0.5 mL at 2×10~5 cells/mL into 5 culture wells respectively,and 2μl liposome was added for each well. Five content grads include 0.4μg,0.6μg,0.8μg, 1.0μg and 1.2μg were designed. After transfected for 24 hours without serum, transfection liquid was removed. Finally we got the results of the optimal DNA contents were 0.6μg,0.4μg, 1.0μg and 0.6μg for BmN-SWU1,BmN-SWU2,BmN and Sf21,respectively.2. Optimization of liposome content.We inoculated 0.5 mL at 2×10~5 cells/mL into 5 culture wells respectively,and 0.6μg DNA plasmid was added for each well.Five volume grads including 1.0μl,1.5μl,2.0μl,2.5μl and 3.0μl were designed.After transfected for 24 hours without serum, transfection liquid was removed. Finally we got the results of the optimal liposome content were 2.5μl,1.5μl,3.0μl and 3.0μl for BmN-SWU1,BmN-SWU2,BmN and Sf21,respectively.3. Optimization of cells density.We inoculated 0.5 mL cells suspend of BmN-SWU1,BmN and Sf21 at 0.5×10~5 cells/mL,1×10~5 cells/mL,2×10~5 cells/mL,3×10~5 cells/mL and 4×10~5 cells/mL respectively into culture wells. When came to BmN-SWU2, We inoculated 0.5 mL cells at 1×10~5 cells/mL,2×10~5 cells/mL,3×10~5 cells/mL and 4×10~5 cells /mL,5×10~5 cells/mL respectively into culture wells.0.6μg DNA plasmid and 2μl liposome was added for each well.After transfected for 24 hours without serum, transfection liquid was removed.The optimal cellular density we got were 1×10~5 cells/mL,5×10~5 cells/mL,2×10~5 cells/mL and 0.5×10~5 cells/mL for BmN-SWU1, BmN-SWU2, BmN and Sf21,respectively.4. Optimization of co-transfection time. We inoculated 0.5 mL cells at f 2×10~5 cells/mL into 5 culture wells respectively, and 0.6μg DNA plasmid with 2μl liposome was added for each well. Transfected without serum, then removed the transfection liquid after 6 h,12 h,18 h,24 h and 30 h respectively.Finally we got the results that the optimal co-transfection time were 24 h,6 h,24 h andl2 h for BmN-SWU1,BmN-SWU2 and BmN,Sf21,respectively.5. Optimization of serum content in transfection liquid.We inoculated 0.5 mL cells at 2×10~5 cells/mL into culture wells respectively,Non-serum and 5% serum were designed for transfection liquid of BmN-SWU1,BmN and Sf21. When came to BmN-SWU2,0.5 mL cells at 2×10~5 cells/mL into culture wells were inoculated separately. five serum content grads including 2.5%,5%,7.5%,10 % and 12.5 % were designed for BmN-SWU2. 0.6μg DNA plasmid with 2μl liposome was added for each well.After transfected for 24 hours without serum,transfection liquid was removed.Finally we got the results that the optimal serum were zero,5%,zero and 5% for BmN-SWU1,BmN-SWU2, BmN and Sf21, respectively.6. BmN-SWU1,BmN-SWU2,BmN and Sf21 were transfected on the optimal condition, and the highest transfection efficiency were 55.08 %,8.29 %,2.0% and 11.66%, respectively.7. BmN-SWU1 which had the highest transfection efficiency was transfected and then filtered by G418.The G418 lethal curve of BmN-SWU1 showed that 300μg/mL G418 was the minimal lethal dose to BmN-SWU1.Transgenic cells were filtered by 400μg/mL G418 for 14 days and finally we got a cell colony with over 60% transgenic cells.Transgenic cells colony was filtered by 200μg/mL G418 for about five months, and we got a cell colony with over 95% transgenic cells.By far,the transgenic cell line has been subcultured to the 15th generation, and could be subcultured every six to eight days, which corresponds to the subculture time of non-transfected BmN-SWU1.The transfection conditions had been optimized during the process of the cation liposome lipofectamine 2000 transfecting BmN-SWU1,BmN-SWU2,BmN and Sf21,the highest transfection efficiency of BmN-SWU1 were 55.08%.We got a cell line with over 95% transgenic cells, and successfully constructed cation liposome transfection system, which lay the foundation for analyzing the transgenic vector expression of silkworm, establishing silkworm engineering cell line and raising transgenic silkworm.
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