Dear all,
I'm working with lentiviral vectors and aiming to overexpress my gene of interest in CHO-k1 cell lines.
I have tried several protocols to reach the high titre of the virus, however the efficiency of transduction is quite low since there are very few green cells and GFP light is pale green; my crowning achievement !!
Finally I decided to check transduction repetation .I'm going to select transducted cells after antibiotic screening and infect them again. so here is my question, is this method beneficial or not?
any comment or suggestion in this regard will be highly appreciated.
Thank you in advance,
Hi Niloufar,
From my knowledge CHO-K1 cells should be very easy to transduce. Check this paper:
Article Evaluating the effectiveness of transferrin receptor-1 ( TfR...
So regarding the virus:
The quality of virus is very important
What is the titre of your virus?
What MOI are you using (paper suggested MOI of 100)?
Are you using ploybrene?
What promoter are you using (is it CMV?)
Sponiculation also helps so have you tried it?
Reboosting with virus might help but if the quality of the virus/transduction is not good enough I do not expect to see a substantial increase in GFP expression
Another way is to do the selection and perform cell sorting to select the cells with highest GFP expression, or you can do monoclonal selection (choose the clones with highest GFP expression)
I hope this helps.
yes you may repeat infectionto obtain higher efficiency I tried it before and it worked.
hi
viral particle quality and concentration is critical for cell transduction >>
you can used these kits for your project :
10-minute, quantitative lentiviral titer test—Lenti-X GoStix Plus - Clontech
Lentivirus purification kits - Clontech
other choice , you can order ready viral particle :
https://www.genecopoeia.com/product/premade-lentivirus/?utm_source=genecopoeia.com&utm_medium=display&utm_campaign=also_viewed
http://www.amsbio.com/lentivirus.aspx
good luck
Yes you can do it. I have done 3 infections every 2 h for 6hours.it improved. U can sort them using facs and propagate before analysing ur protein of interest.
- Badges
- Science method