Hello again,
I have another question about silencing.
- I did transfections with 4 different sequences (using shRNA plasmids) for the same target in a cell line.
- After 3 days I trypsinized the cells and re-plate them on selective medium (plus G418, concentration determined previously).
- After 3 days of culture all non-transfected cells were killed and after 4-5 days I trypsinized the "polyclonal" cells for subculture at low confluence and saved an aliquot to analyze target mRNA levels.
- Two of the plasmids had a 60-70% reduction in mRNA expression. I chose these cells to tray to obtain monoclonal cell lines of stable transfected cells.
- For that, Iused 2 methods:
a) Limit dilution in 96 wells plates.
b) Manual colony pickup
Method a): I have not enough cells yet to analyze expression.
Method b): I analyzed five clones and they show no reduction in mRNA compared to control clones!
What may be the reason of these results if the cells are able to grow in selective medium?
The shRNA sequence is not effective? Any other reason?
I will appreciate any help/advice with this.
-anahifranchi-
shRNA can be tricky. The main thing is you do not know where the shRNA will intergrate into the genome. It is possible that the shRNA is integrated into inactive heterochromatin or recombination damaged the shRNA construct. This would maintain antibiotic resistance, but hinder shRNA expression. It is also possible that something is altering the formation of your shRNA. When you ordered the lentivirus, did they give you an information sheet with binding information? Sometimes this costs extra, but it would allow you to check possible binding sites of your shRNA.
Alot of things could be going wrong. It is hard to pinpoint just one.
Did you use a negative/positive control?
Did this help?
-jerryshelly1-
Thanks jerryshelly for your response.... here I come with some more info and questions...
The plasmids are constructed on demand for a custom target gene. The company designs the sequences (4 targets and 1 scrambled) and inserts them into the palsmid. Four different sequences located on the same site of a parental plasmid. Is the Sure Silencing shRNA plasmid kit system from QIAGEN.
Beyond that, in any of the options you mentioned above, the events do not occur in the same way in all cells ... ie, some cells should the plasmid integrated into an "convenient" site. In fact, the analysis of the first subcultures shows reduced levels of mRNA for the target gene.
So, as I understand there is a proportion of cells expressing the sequence into an appropriate site of the genome. If this is true, it means that increasing the number of clones analyzed I should be able to find at least one clone that shows silencing ... ?
My controls: I compared the 4 sequences against a random sequences also provided by the kit. In addition I included a sample of non-transfected cells .... that in fact shows higher expression levels ...
-anahifranchi-
You should be able to. I have always had problems with shRNA expression when the passage becomes to high. I like using a GFP tagged contstruct to determine the effectiveness of the shRNA. Your method is fine though.
-jerryshelly1-
