Trouble with shRNA transfection - (Jun/17/2013 )
I need some help about shRNA.
Here is my story : I ordered shRNA plasmids from a commercial firm ( don't know if I can tell the name without being kicked of the topic...) to invalidate the receptor I'm studying. I made amplification and purification, then digestion to linearize the plasmid and transfected it in the cells.
After 24h-48h of rescue, I started a selection with puromycin at the minimal letal dose (I made a killing curve before, and obtained 0,1 µg/mL for one cell line and 1µg/mL for another). I had a massive death of the cell during one week, then growth of resistant clones.
I made a FACS analysis to compare the "target sh" versus Non" target sh" cells.................. and they have the same rate of expression, i.e no invalidation....The cells grow well in puromycin....
Does someone ever face the same problem?
I just see two possibilities : either I had no luck and selected "natural" resistant clones in the culture, which do not integrate the plasmid or the shRNA is not efficient (but I think the firm has validated the sh.....)
How many different shRNA plasmids did you use to try to eliminate expression of your gene. Sometimes the plasmids do not knockdown your gene of interest to the levels the manufactures would expect. Puromycin is a powerful mammalian selectable marker. I doubt your cells are resistant to it without your plasmid being present, especially sense you saw complete death with a kill curve. Are there multiple copies of your gene? Were you able to obtain the sequence they used for the shRNA. Sometimes you have to pay extra for this service, but it may help in determining what is happening in your cells.
The kit is made of a pool of 4 different shRNAs plasmids (I transfected 5 µg for 5 millions of cells, with the same quantity of each plasmid), but I do not have the sequences (I will ask the manufacturer). There should be only one copy of my gene but at least one of my cell lines is polyploid, but I did not checked my locus of interest (will look after this too).
Thank you for your answers.