280016 Precise Gene Delivery for Cell Reprogramming with Nanochannel Based Electroporation (NEP)
Cellular reprogramming has presented great promise for regenerative medicine and tissue engineering. Pluripotent stem cells can be generated from mouse and human somatic cells by enforced expression of defined factors. However, clinical uses of current virus methods were limited by integration of transduction factors to the host genes, which may result in tumorigenicity. Meanwhile, even current nonviral methods successfully avoid exogenous DNA integration, low transfection efficiencies and poor reproducibility are still main problems to be solved.
Our lab developed a nanochannel based electroporation device to deliver trancription factors, which may achieve dosage control and minimize side effect of electroporation and result in high reprogramming efficiency and quality.
In this study, mouse embryonic fibroblast (MEF) cells were loaded into microchannels via rotation. The pCAG2LMKOSimO vector containing complementary cDNAs of Oct4, Klf4, Sox2, c-Myc and m-Orange was introduced into mouse embryonic fibroblasts (MEFs) by NEP method under various conditions. Expression level of transduction factors was followed and characterized by image analysis and RT-PCR method. High percentages of cells expressed red fluorescence after 24h of cultures, which indicated successful transfection of pCAG2LMKOSimO plasmid. Compared to bulk electroporation method (Neon, invitrogen), MEF cells transfected by NEP method also showed longer red fluorescence expression time and more homogeneous red fluorescence distribution which indicated longer gene expression time and gene delivery dosage control.
Our newly developed nanochannel based electroporation (NEP) technique was used to transfect MEFs with the pCAG2LMKOSimO vector and achieve a successful precise dosage control. The results greatly suggested NEP a potential alternative delivery method which may increase cell reprogramming efficiency and avoid tumorigenicity.