A method often used by researchers in the field of molecular biology is the controlled transport of external DNA or RNA into eukaryotic cells. This so called transfection uses the services of plasmids, transposons or viruses to incorporate the DNA/RNA into the cell genome and allows the analysis of the regulation and expression of genes or the production of recombinant proteins.
Transfection Methods
The transfection of DNA can be either transient or stable. When performing a transient transfection the expression of transfected genes in cells is usually measured within 16 to 96 hours. However a stable transfection integrates the DNA into the genome of the cell and therefore allows unlimited analysis of the gene function. One also can transfect several genes in parallel with the co-transfection method.
There are various methods of introducing DNA into a cell, for example chemical or physical methods. The chemical transfection uses calcium phosphate, DEAE-Dextran liposomes, organic polycationic polymers and dendrimers whereas the physical transfection is either done by microinjection or electroporation.
Requirements for Transfection Reagents
Transfection reagents differ considerably in transfection efficiency. Most reagents transfer DNA inefficiently as cells only take up particles of a specific size. In addition, most reagents cannot sufficiently protect the DNA-complex against endonuclease degradation. New carrier molecules with new chemical features allow the DNA-complexes to be taken up more efficiently, thus increasing the transfection rate. Another issue that comes up frequently when choosing among different gene delivery methods is the cytotoxicity of the various reagents.
Selection Antibiotics
For the differentiation between transfected and non-transfected cells, researchers use selection-markers which are co-transfected with the external DNA. In many cases they use resistance genes for antibiotics like the neo-gene which encodes a resistance against Geneticin. Other selectable markers such as Puromycin, Hygromycin B, Blasticidin or Carbenicillin are also frequently used.
PAA's Product Line
Based on principles of nanotechnology PAA developed a new transfection reagent. Nanofectin is taken up by the cells in a very efficient way and protects the bound DNA against endonuclease activities. Nanofectin consists of two components, a porous nanoparticle and a positively charged polymer which encases the nanoparticle. The complex easily passes the core membrane and the external DNA is integrated into the eukaryotic cell DNA. Nanofectin can be used for adherent as well as non adherent cells and can also be used for stable and transient transfection.