Abstract
In this study, we developed a new purification method using chondroitin sulfate C (CSC) and protamine sulfate (PS) to concentrate lentivirus. To evaluate the efficiency of this new method, we compared it with several previously described purification protocols, including virus concentrated by ultracentrifugation (Ultra), precipitated by polyethylene glycol (PEG), and sedimented by CSC combined with polybrene (PB). After using the different methods to purify and concentrate equivalent amounts of lentivirus supernatant, the virus pellets precipitated by the different methods were resuspended using the equivalent volumes of DMEM. Subsequently, 10 μl of each lentivirus stock carrying EGFP gene was used to transduce two types of cells, human embryonic kidney 293T (HEK293T) cells and mouse mesenchymal stem cells (mMSC). It was obvious that HEK293T and mMSC appeared much intensiver green fluorescence through virus transduction from PS method than from other methods. To quantitate the transduction efficiency of the viruses, we examined virus titer in the cells after transduction using a real-time PCR-based analysis. Accordingly, we verified that PS precipitation could generate virus with a higher titer (4.39 × 108 IU/ml) than PB (2.43 × 108 IU/ml), Ultra (1.16 × 108 IU/ml), and PEG (0.56 × 108 IU/ml) in HEK293T cells. As for HEK293T cells in mMSC, the PS method also generated virus with a higher titer (4.66 × 108 IU/ml) than the Ultra method (2.36 × 108 IU/ml), and a much higher titer than those of the other chemical-based precipitation methods using PB (4.82 × 106 IU/ml) and PEG (8.98 × 104 IU/ml). Furthermore, the HEK293T cells and mMSC transduced by PS(1X)-virus appeared to have higher cell growth ratios, respectively, than the HEK293T cells and mMSC transduced by lentivirus using the other methods. We conclude that our new method for purifying lentivirus is cost-effective, time-saving, and highly efficient, and that lentivirus purification by this means could possibly be used to transduce a variety of cells, including stem cells.
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This study was supported by grants from the National Science Council, Taiwan (NSC 104-2321-B-415-001-).
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Lee, JY., Lee, HH. A new chemical complex can rapidly concentrate lentivirus and significantly enhance gene transduction. Cytotechnology 70, 193–201 (2018). https://doi.org/10.1007/s10616-017-0133-0
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DOI: https://doi.org/10.1007/s10616-017-0133-0