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lentiviral shRNA MOI questions


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#1 spinietzschon

spinietzschon

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Posted 09 March 2011 - 09:21 AM

Hello everyone,

I have 2 basic questions and one less important question afterwards regarding MOI.

Background - chordoma cells which grow slow are being transduced w/puromycin resistant and anti-T (transcription factor) shRNA and we're not trying to get a monoclonal growth but rather a stable population (mostly on account to the very slow doubling time).

Using a MOI of about 1 virus unit to 10 cells (we can waste cells; but the virus is expensive...), versus 5 virus to 10 cells, seems to not have gotten me more transduced cells at all. So I tried 1 virus to 20 cells and it got the same results roughly. Also, before this we had used a MOI of 1, and 5; each seemed about equally good with each other and with the lower MOI's.

Questions:
1) Is there likely just a (low %) set proportion of cells in any population of mine amenable to transduction with this vector and I should just go for a low dose as it's just as good as a high dose, to save lentivirus?
2) Relating to (and possibly contradicting #1...) - So, say that normally one virus transducing into a single cell and integrating knocks down T expression 90% (10% normal remaining). If it's infected twice, would it (just making up #'s obviously) say knock it down to ~1-2% normal expression, or are vectors normally made to express so highly that infecting 2x wouldn't do anything that the first infection does? (Or, does this have to be figured out with each vector and each cell line experimentally?) .... In essence I'm saying, even if the actual # of cells transduced are not increaing with more MOI, can there be an effect we would want to be aware of and look into, or should we just use the low MOI to save materials given the circumstance?

Thank you very much. Again, PLEASE only answer if you have a good source or personal experience with lentiviral shRNA - everyone around my lab is good at reasoning what 'could' be the case but I'm joining this forum hoping to get ahead a little bit! Thank you!

ONE more thing: I expected the TF to stay in the nucleus as... It's a TF. But, the nuclei of the controls (w/scrambled sequence and GFP) are only in the nucleus as well. Is there a reason they're all local? I was assuming the 'scrambled' sequence would not code for them to hang out in nucleus i.e. I figured they'd be in cytoplasm as well etc but they're only lighting up that nucleus. Uh... Not a big deal just wondering. The actual prodcut # if that's helpful is santa cruz, sc-10804 - http://datasheets.sc.../sc-108084.pdf. Thanks!

Edited by spinietzschon, 09 March 2011 - 09:27 AM.





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