I had the same identical problem. I established 4 stable cell lines, each over-expressing shRNA against my gene of interest. Every shRNA targets different sequence on mRNA of my gene of interest. The selective marker, GFP, was successfully expressed in all 4 cell lines and only green cells were selected by Flow Cytometry. No gene knock-down was detected in any of these cell lines. I think, if the marker is expressed, the absence of knock down could not be attributed to the region of construct integration. However, I also did not find any explanation to this problem.  

I am not much of a Molecular Biologist, but I think these are good questions which may not be completely explained in the field.

These days, when working with RNA interference, it is a general practice using Lentivirus vector to establish stable transduction of a cell to deliver specific shRNA for consistent expression, and which can completely knockdown expression of the target gene. It is not rare or unusual for researchers to find that the cell is resistant to puromycin but does not result in gene knockdown. “Why” is the question asked? There are different some plausible explanations, for example, promoter activity differences, the terminator of the upstream gene may not be efficient enough etc. However, in this specific case, 4 clones did have gene knockdown and 6 other clones not.

The good thing about this is that the investigator found that the 4 knockdown clone integrated into intron region of some genes and the 6 failed knockdown clones integrated into non-gene areas. It is well-known that if integration of a gene into inactive area, or non-gene area of a chromosome, the expression level will be very low,  and since the chromosome has more inactive area than active area, more clone will be found integrated into inactive area of the chromosomes. By improved selection of higher expressed clones which mainly integrated into gene expression active area of a chromosome, industry can easily produce protein in CHO cells to 10-20 grams/liter of culture medium for target protein. So, in this case, one could guess it may need relatively low level of expression to maintain puromycin resistance, but much higher level of shRNA may be required to maintain knockdown of a gene. To prove if this guess is true or not, the investigator could do parallel culture of the 4 knockdown clones and 6 failed knockdown clones by serially increasing the concentration of puromycin in the culture medium. If the guess is right, at certain concentration of puromycin, the 4 clones should still grow well while the 6 clones will die due to not-enough expression of the puromycin resistant gene. I am in no way an expert in this area.

Just consulting and reading. I hope this is helpful.

Hi,

Depending of two things:

- Regions of the integrations sites (active or not for the transcription)

- Number of integrations in the clone

These two parameters influce directly the level of expression and so the level of inhibition by sh-RNA expression.

If you want enhance your pourcentage of puroR clones which knock down your target, I advise you to test inferior puro concentrations. However, 4 positives clones is not too bad ;)

Nicolas