Same backbone different GFP intensity - (Nov/05/2010 )
I am using pGIPZ miRNA system which has GFP on it. When i transfect Non-silencing (control) or the different miRNA plasmid clones into HEK293T cells to verify knock down of the gene of interest, I found that the GFP fluorescent of two of my miRNA clone were significantly lower than the non silencing control. I have 3 different plasmid clone targeting different sequence of the gene and only 1 has similar intensity to the non-silencing control. I have double checked the concentration hence quite certain that same amt. DNA was transfected.
Does this usually happened (clone variation?) But the GFP are driven by common mammalian promoter? Or it's a technical glitch happening here??
I have cloned several genes into pGFP vectors, and I have noticed the longer the insert, the lesser the GFP intensity. my boss says it is because of distance to the promoter!
The miRNA precursor sequence cloned on the pGIPZ vector is one the same "exon" (or transcript). So, when the pre-miRNA gets processed, the GFP mRNA will be degraded. More of pre-miRNA gets processed, less GFP will be expressed.