Hi
I did transfection using lentivirl cas9 to generate cells expressing cas9. I used balstodzen antibiotic as a marker for my transfection and everything went well. However, when I did western blot to investigate the expression of my gene of interest, I noticed reduction in the expression!!!!!
these cells suppose to be a negative control for my experiment.
so Iam not sure why the gene expression is reduced comparing with the wild type cells ?
Any suggestions I will be appreciated
even negative cell under the antibiotic selection and going through some kind of stress. Just use the negative cell rather than wild type as control, or both. sometime pick up several lines for each treatment will be more convincing.
Could you elaborate on what you mean by doing a western for your gene of interest? Did you do a western with AB's against the Cas9 you wish to integrate into the genome or another gene? Also did the Cas9 you integrated via lentiviral vector come with the antibiotic resistance and a sgRNA scaffold coding for a sgRNA targeting your gene? Could you also explain what viral vector and helper plasmids you used to package the lentiviral plasmid as well?
Did you also verify the insert site to rule out the unlikely event that the virus integrated in an important site relating to said gene. Again unlikely but you never know.
You can use both of them. and later you can compare to them, Whether the expression reduce or increase .
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