Hi All
I would like to know if any one knows the knockdown cell line with lentivirus. I mean I generated the knockdown cell line with lentivirus and the cells were selected by puromysin for many day. The uninfected cells were all died. I do not know if I could use this cell line for my experiments all the time.
Thank you so much.
Best
Hi Lei,
I don't really know what is your question, since your selection process looks fine.
If you validated your KD by WB, then you can spare some cells for your stock... and use them for years...
Try to keep them under selection when they are in culture of course.
Best
Yes, you can proceed to further characterize the cell line if the cells are resistant to puromycin. Listed below are certain things that you need to consider before concluding the effects of knockdown:
1. First, you can not compare the phenotype of the knockdown cell line with a No lentivirus normal cell line simply because you have no virus and as well as no puromycin resistance or exposure to the antibiotic in the normal control cells. You need to have a separate shRNA (non-targeting) control. You need to develop the control cell line at the same time as the knockdown cell line. These cells should also receive the same concentration of puromycin as well and also should be maintained in parallel to the same passage number for the same time period as the knockdown cell line in order to have reliable results of knockdown.
2. Remember the knockdown in combination with the selection procedure takes a long time and duration that the effects you observe are most likely to be chronic effects rather than any acute change due to knockdown. This will include some compensatory mechanisms that have arisen due to the loss of target. Therefore, it is unlikely that you can reverse the effects of knockdown by overexpressing the same gene at a later time due to the effects of compensatory mechanisms that are not directly influenced by the gene and make a toll in masking the reversal.
3. The whole transduction procedure and stable cell line development including the control needs to be verified in independent experiments to check the reproducibility.
You can also use the CRISPR system in "all in one" lenti with rporter gene (puro, GFP....)
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