Byun J, Kim J M, Kim S H, Yim J, Robbins P D, Kim S
Institute for Molecular Biology and Genetics, Seoul National University, Kwan-Ak-Gu, Korea.
Gene Ther. 1996 Nov;3(11):1018-20.
We have developed a simple and rapid method for the determination of retroviral titer by G418 selection which is both accurate and reproducible. NIH3T3 cells were transduced with recombinant retroviral vectors harboring the neo gene and selected in the presence of the antibiotic G418 for 3 days. Cells were then trypsinized, harvested, and counted on a hemacytometer. The control NIH3T3 cells were completely dead 3 days after treatment with G418, while cells transduced with retroviral vectors containing the neo gene produced an average of 15 progeny cells per colony. To estimate viral titer, therefore, the total number of trypsinized G418-resistant cells was divided by 15. Retroviral titers measured by this method did not differ significantly from those determined by the conventional method based on counts of G418-resistant colonies 10-14 days after G418 selection. The method worked well with various retroviral constructs and its efficacy was not influenced by cDNA insert. This method not only reduces the amount of work but also shortens the time needed for determining viral titer.
我们开发了一种通过G418筛选来测定逆转录病毒滴度的简单快速方法，该方法准确且可重复。用携带neo基因的重组逆转录病毒载体转导NIH3T3细胞，并在抗生素G418存在的情况下筛选3天。然后用胰蛋白酶消化细胞，收获细胞，并在血细胞计数板上计数。用G418处理3天后，对照NIH3T3细胞全部死亡，而用含有neo基因的逆转录病毒载体转导的细胞每个集落平均产生15个后代细胞。因此，为了估计病毒滴度，将胰蛋白酶消化后的G418抗性细胞总数除以15。用这种方法测得的逆转录病毒滴度与基于G418筛选后10 - 14天G418抗性集落计数的传统方法测得的滴度没有显著差异。该方法对各种逆转录病毒构建体均有效，其效力不受cDNA插入片段的影响。这种方法不仅减少了工作量，还缩短了测定病毒滴度所需的时间。
