RNAi side effects - (Apr/10/2008 )
Hello everyone,
How do you exclude the possibility of siRNA side effects?
I knockdown a gene by shRNA expressing lentivirus. The KD was efficient, and the cell morphology was changed. But later I found that this morphologic changes were due to the side effects. Because ectopic expression of a RNAi escaping mutant did not rescue the cells. Besides, the shRNA had similar effects on cell lines from different species.
You may want to blast the sequence of your RNAi and see if it could target any other genes. If so, you may very well knockdown another important gene.
If the blast do not give any specific answer, I would try go design anoter shRNA.
The problem may also come from the virus. Too many viruses can cause cellular responses, such as change in morphology. Have you tried infecting your cells with different virus concentrations?
If the blast do not give any specific answer, I would try go design anoter shRNA.
The problem may also come from the virus. Too many viruses can cause cellular responses, such as change in morphology. Have you tried infecting your cells with different virus concentrations?
Thank you Madrius,
I used lower virus concentration and the problem remained. Strangely, different shRNAs had the same effects, i.e. induction of the formation of multi-nucleated giant cells.
And all of your constructions give the same phenotype as the empty vector?
If so, your virus itself could very well be the problem..
If so, your virus itself could very well be the problem..
No, only 2 of the 5 tested lentivectors caused the phenotype. And the phenotype was not related with the degree of knockdown.
After several passages, the phenotype remained, so the effects were likely genetic.
Wow, this is weird!
Have you repeated the experiments with the same viruses that cause the phenotype more than once?
Lentivirus sometime integrate their sequence into important genes, wich then lead to a weird phenotype.
Have you repeated the experiments with the same viruses that cause the phenotype more than once?
Lentivirus sometime integrate their sequence into important genes, wich then lead to a weird phenotype.
Yes, I repeated it. If it were the case of insertional mutagenesis, there should be only a small portion of the cells having that phenotype. However, my cells seems quite homogenous.