I have a question.
We are doing transient transfection, shouldn't we use mRNA instead of DNA? because we do not want our fragment to be incorporated in the genomic DNA of host cell? isn't that the point of transient transfection?
mRNA is transient (short-lived) so shouldn't it be used for short term incorporation? also mRNA only needs to reach till cytoplasm as it can translate outside nucleus?
Which one is better DNA or mRNA transfection?
It depends how short of a time window you want the cell to express your protein for. Plasmids that are non-replicating can also be used for transient expression with a low chance of genomic incorporation though homologous recombination. You should not be using a transposon based system if you only want transient expression.
Sorry for being a little repetitive.
Shouldn't mRNA be better for transient because it does not need to go to nucleus and can leave our gene of interest literally anywhere in the cytoplasm and after translation, it should disappear. Whereas in generating stable pools, we need our gene of interest to be integrated into the genome of the host.
Also, transposase are short lived. Do they not deliver our gene of interest into the cell and wither?
Plasmids can persist in the nucleus transiently without integrating into the genome. They typically can persist for days to weeks. With mRNA it is mostly degraded within the first 1-4 days and never lasts more than 7 days. As I stated the choice to use a plasmid or mRNA is mostly down to the length of time you want the transient expression for.
Transposases (originally from transposons which are also known as jumping genes) are used to transfer the section of the plasmid between the ITRs into the host genome for integration and stable expression.
- Badges
- Science topic