For several months now, I've been attempting to generate and infect my cells with lentiviruses, but unfortunately, I have encountered persistent challenges. The specific cells I aim to transduce are human fetal fibroblasts (HFF), and the plasmid I'm using is "dCas9-5xGCN4-P2A-BFP" with Addgene number 184438. Interestingly, this plasmid has demonstrated success in transducing other cell lines for different research groups. However, after thorough investigation, I have ruled out the cells themselves, our protocol, and personal error as potential causes for the unsuccessful transduction.
It's worth noting that I have successfully created lentiviruses and transduced HFF cells with high efficiency using other plasmids. Despite my efforts, the lentiviral concentration and the spinoculation method for transduction have not yielded positive results.
If you have any insights or advice to offer regarding this issue, I would greatly appreciate your input.
I didn't determine the titer, I have tried the concentration and spinoculation both seperatly and together but non of them worked.
Ok thanks for the information. I suspect, given the large size of dCas9-5xGCN4-P2A-BFP, that the RNA is not being efficiently packaged into the virus. The larger the RNA, the less efficient packaging is, resulting in virus particles with no nucleic acid in them. I have experienced this previously and the amount of lentivirus produced/titer was extremely low resulting in unsuccessful transduction of the target cells.
You may want to contact the research groups that had success in transducing this plasmid and find out how they produced their lentivirus.
Good luck!
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