So this is more of a curiosity of mine. Every protocol I find about the transfection of T-lymphocytes includes a two days stimulation prior to the transfection and I am wondering why that is. Can't the stimulation and transfection occur at the same time?
In order for the transfected DNA to be taken into the nucleus it requires the nuclear membrane be broken down. Thus the cellular division (G2->M) is required to integrate the transfected DNA into the nucleus for proper expression and processing.
This is one of the major caveats of gene therapy and the reason adeno/lenit-virus are used for gene transfer. They have the capacity to integrate into the host genome of terminally differentiated (non-dividing/slow dividing) cells.
I guess It depends on the stimulus... I used to transfect them and then stimulate
In order for the transfected DNA to be taken into the nucleus it requires the nuclear membrane be broken down. Thus the cellular division (G2->M) is required to integrate the transfected DNA into the nucleus for proper expression and processing.
This is one of the major caveats of gene therapy and the reason adeno/lenit-virus are used for gene transfer. They have the capacity to integrate into the host genome of terminally differentiated (non-dividing/slow dividing) cells.
David is right. In any cell type the best time for uptake of DNA is when cells are dividing. Depending on the cell type and division rate, one can make an educated estimate of when to transfect. Two days after stimulation for T cells is perhaps when they are mostly dividing.
We are using T cell line and for transfection we use electroporation. In this process there is no need of prior activation of T cells.
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