Quite a naive question
I am looking for the most optimal way to transfect different cell lines with the same construct. Some of them are notoriously difficult to transfect, like RWPE1
I am satisfied with the quality of transient DNA transfection, which I did on simple lines like HEK293 or HeLa. But maybe it’s time for me to somehow optimize the process? Please advise, maybe it’s time for me to learn CRISPR? pLenti? Something else?
talk to me please
I would recommend before the optimization process to check whether your cell lines have Mycoplasma. They are a subtle contamination that doesn't behave like your normal run of the mill bacterial & fungal contamination with cloudy apperance & pH changes to the medium. Mycoplasma are a known source of inhibitor, I've dealt with stocks that were positive and they reduce transfection efficiency greatly. Nobody thinks they have Mycoplasma until they check it :).
Since your construct works in HEK293, I recommend that you try to optimize lipo transfection conditions first, actually I am working on LnCaP, and the transsite transfection system works well using the lipo3000 system. Alternatively, electroporation can be tried if necessary. The Lenti way takes time, unless you have no choice. If your goal is to KO, KI, or generate mutations, you may want to try the RNP system, it works well in our hands.
It is difficult to have an single optimal way for every cell lines, each cells have their own strength driving multiple signaling pathway, upper limit of the plasmid copy received and interaction between GOI and its own proteins, hence affecting gene expression facility. We had tried at least four different transfection method with more than ten different reagents just to make a cell population to express 0.1% of GOI, while in 293T the expression was almost hitting the upper limit of detection in flow cytometry. Therefore, to generate an optimized protocol for each cell lines or primary cells are considered easier then to generate a single protocol for universal use. The cloning process or to achieve a single double KI/KO clone by CRISPR is very time consuming and troublesome, if you want to go through CRISPR, try look at the newest technique such as base/prime editing by Dr. David Liu. In the other hand, lentivirus is not, in our lab, lentivirus is an useful tool for large scale cell line production, a single production process may grant us enough amount of lentiviral particle for more then 100 times of usage. However, if a gene is already being difficult to express in such cell lines, I would rather look into an optimization of DNA construct, a small twist such as codon optimization or driving promoter sometimes grant you more then 50-fold of difference, that will be far practical then transfection process.
Best
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs