j********r
发帖数: 156 | 1 最近一直在尝试overexpress a gene in primary epithelial cells, but always
failed to get decent level of expression。望版上诸位帮看一下。
我用的是一个类似与pBabe的retroviral vector, the gene was tagged with GFP or
Flag on the c-terminal. The target cells were infected twice and then
subjected to antibiotic selection (blasticidin in this case). 用anti-
Flag做western, 勉强能看到带，如果用the ab against itself, 可以看到exogenous和
endogeneous的差不多 （the endogenous level is already very low, which is
also the reason that we hope to increase it to hopefully make some
phenotypic observation)。
我的问题是，是不是retroviral表达系统本身不会达到很高的蛋白表达 (say if
compared to
transient transfection plus selection， if the cells are not hard-to-
transfect)? It is also possible that the moi is low in my hands
therefore causing low copy number of transgene per cell.
多谢！ |
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