I've sub-cloned M-MITF in a lentiviral system for over-expression and I'm having a really hard time detecting its over-expression despite >95% of cell surviving puromycin selection (and I'm sure the puromycin I'm using is working: it kills the uninfected control very efficiently and fast). I can get some low-level over-expression within the first few passages but then it pretty much disappears, despite constant selective pressure (I always use a positive control on my blots to make sure the antibody works properly). I used the same system to clone at least another dozen genes and always had beautiful signals, so I was wondering if there's anything peculiar to M-MITF that I'm not aware of? FYI I'm using HEK293 for packaging and various melanoma cell lines for the infection.
Thanks!!
What promoter do you use to control the expression? Do your melanoma cell lines express MITF at all? Or do they use RNA based silencing (like miRNAs) to downregulate the MITF expression? Did you confirm the integration of your construct by PCR/sequencing?
Thank you so much guys!
Yes I did confirm by sequencing the insertion and correct sequence. I'm expressing it in both cells that have some basal expression and some that don't, as well cells of different lineage (BRAF vs NRAS mutations)....in all I see a little overexpression at first but then it tapers off very quickly....still trying to trouble shoot.
Thanks again,
A.
It is cytotoxic at high levels, but you should have no trouble overexpressing it with lentivirus. I have no trouble expressing M-MITF by lentivirus.
Hm, thats more tricky. I have used plasmid based transient overexpressions when I needed to overexpress this in different cell lines (melanoma and non-melanoma) as well as lentivirus. This worked both without problems. Which promoter are you using to control your insert?
Let's break the problem down into sequence design, transduction, transcription and translation:
Double-check the appropriate expression elements in your design: Kozak context, unwanted secondary structures etc.
It's clear that your cells are efficiently transduced (>95% cell survival).
You should check the transcription with RT-PCR and if there isn't an efficient transcription, you should go through the promoter and lentiviral inactivation issues: Although the promoter of puroR is working, you need to check the promoter of M-MITF. Some so-called ubiquitous promoters are not active in certain cell types. Lentiviral LTRs may also become epigenetically inactive after some passages in some cell types. You can use a GFP expressing lentiviral construct (in the same backbone) to check these issues.
Obviously, if it works for GFP or you have quite high mRNA expression, the problem will be about M-MITF translation. In this case you should check for smicroRNAs or cell toxicity.