Numerous Advantages of the Nucleofector Technology
High Transfection Efficiencies
As DNA transfected by nucleofection can directly enter the nucleus, very high transfection efficiencies can be obtained, even in otherwise difficult to transfect cells such as fresh or immortalized primary cells or many difficult-to-transfect cell lines. Transfection efficiencies of over 50% can be obtained in most cell lines as well as in primary cells. For some primary cells, such as human dermal fibroblasts, efficiencies exceed 90%.
Short Time to Results
As DNA transfected by nucleofection is not dependent on cell division in order to enter the nucleus, the time until transgene expression is significantly shortened relative to other transfection methods. In most cell types, expression of the transfected gene can be analyzed after just 2-4 hours, rather than the standard 24 to 48 hours. This means that experiments can be performed much more quickly, and often completed within a single day.
Simple Method
The Nucleofector technology is based on electroporation, making it a quick method which can be performed in less than an hour. It consists of the Nucleofector device, which delivers the electrical parameters, and Nucleofector Solutions, in which the cells are contained while the electrical program is executed. For each cell type, optimized conditions are being developed in the amaxa laboratories. Consequently, the Nucleofector technology requires minimal optimization (if any) by the user, making it the ideal tool for gene transfer into primary cells, even by inexperienced users.
Fast Protein Expression
As the DNA can directly enter the cell nucleus, expression of the transfected protein can be detected shortly after nucleofection. In cell lines, transfection efficiencies of over 50% can be detected after just 2-4 hours. Even in primary cells, including non-dividing cells, transgene expression can be seen shortly after nucleofection. In unstimulated, resting T cells for example, 15% of the cells express the transfected gene after just one hour reaching up to 70% after 16 hours.
- Early time course of appearance of two different proteins expressed in freshly isolated, non-stimulated human T cells.
Human peripheral blood mononuclear cells (PBMC) were transfected by nucleofection with either the cytoplasmic protein eGFP or the surface protein H-2Kk, a mouse MHC class I heavy chain molecule. Expression of the cytoplasmic protein eGFP can already be detected after 40 minutes, reaching 15% after just 1 hour. Expression of the protein H-2Kk is somewhat delayed as the protein first has to be processed and transported to the cell surface.
