I would like to create lentiviral derived transduced cell lines (neuronal and lung cancer) with overexpression and shRNA knockdown of my protein of interest. As I am going to study mitochondria I thought it would be a good idea to introduce mtRFP in the plasmid and use it as a selection marker for cell sorting. My questions are:
1. What promoters should I use ? ( I would like a strong overexpression of my gene). Will a weak or medium promoter before mtRFP give a signal strong enough for selection? Which promoters are not toxic and effective in both lung cancer and neuronal cells?
2.Is mtRFP overexpression toxic in some way or have any known impact on the function of mitochondria?
3. Are there any additional problems I should be aware during my design?
Hello! (Pozdrawiam serdecznie :-) IMHO all fluorescent proteins are little bit toxic. The stronger expression the bigger toxicity. RFP-s are 'generally' prone to aggregaton so also more toxic than GFP (which is also little bit toxic). Some are monomeric, less aggregation prone but also less bright . Which one of RFP are You going to use? RFP signal frequently overlaps GFP, please look if mCherry or similar will be better.
The promoters may work differently for each cell line but also environmental factors may have influence on level of expression. Nonviral promoter may be preferred. What would You investigate in those cells?
Being a huge fan of fluorescent proteins I would suggest to design also an antibiotic resistance (ex. puromycin which works fast and needs no FACS) vector cause it takes only about 10 minutes to stain mitochondria with one of Mito-trackers.
How do You want to obtain Your construct? Regular cloning or gene synthesis?
http://blogs.nature.com/methagora/2007/04/idiosyncrasies_of_the_human_cm.html
I think that at least dendra2-mito is not very toxic to all kinds of tissue, because there is a mice, Pham-excised, expressing denadra-3-mito ubiquitously. It will be careful when you do long time observation, because photobleaching of fluorescent indicators releases lot of ROS. Roger Tsien has far-red fluorescence protein and images stems cells without any problem. Hope this works for you.
Thank you both!
Adam Figarski--> I am going to investigate mitochondria morphology, short-term live mitochondria dynamics alternations and programmed cell death and oxidative stress after treatment with toxins: 6-hydroxydopamine and Paraquat in neuronal cell line and lung cancer cell line. I will need promoters for overexpression (rather high) both in neuronal cells and lung cancer cells. I have heard that CMV is not good for neuronal cells. Can you suggest some other promoters?
Yes mitotracker is also a good idea and diffrent mitotrackers will allow some additional observations in my established cell lines possible....on the other hand stable cell line with stained mitochondria derived from homogenous transductant is also something nice to have. The best would be to have both but funds are limited.....thank you Chung-chih Lin for suggesting Dendra2 and mentioning the problem of ROS derived from photobleaching - they may have and impact in my studies I have to be aware of this.
I am going to bay custom designed lentiviral plasmids as I am in a hurry and have little time for cloning.
Thank you for your help!
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