Integration events with or without Rep in AAV

• 作 者: Fang Zhu Zhihua Zhang Tiancheng Fan Caihong Yu Liujiang Song
• 机 构: 南华大学医学院 湖南师范大学医学院
• 出 处: 湖南省生理科学会2015年度学术年会暨第十届理事会换届选举大会
• 发表日期: 2015年9期

In this study, we wish to see if we can isolate the transfected cells from untranfected one and to test the integration efficiency of rAAV/GFP with or without rAAV/Rep, as well the efficiency to form large colonies in the single cell culture condition.Six days after cloning, on the second inspection day,we saw very different colony formation efficiency in different clones.Some clone show no expansion at all or very limited expansion.In these colonies,only single cell or very few cells can be seen in the well.While some other colonies expanded tremendously.In these wells, the single cell placed in the well 6 days ago, grown into more than 50 cells.A couple of wells had around 80 green cells present.That is more than doubled 6 times from a single cell.From these data, we can calculate that some K562 cell can double in less than 24 hours.We made an assumption that the virus genome in these cells are integrated.We argue that after all of the cell dividings, if the viruses remained epsomal in these cells, the intensity of the GFP fluorescent would be reduced dramatically.Base on this assumption, we used the number of wells that containing green cells at first inspection, which was three days after placing the single cell into a well, as the number of cells that were successfully transfected.After culture for long time, our data show that AAV with Rep had much higher integration events that the other groups.