Generation of magnetic nonviral gene transfer agents and magnetofection in vitro.
This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3-5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.
Mykhaylyk
Olga
O
Institute of Experimental Oncology, Technische Universität München, Ismaninger Strasse 22, Munich 81675, Germany. olga.mykhaylyk@lrz.tu-muenchen.de
Antequera
Yolanda Sánchez
YS
Vlaskou
Dialekti
D
Plank
Christian
C
D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D007501:Iron / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D013778:Tetrazolium Salts / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry; D014162:Transfection / Q000379:methods*
D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; D004247:DNA; D007501:Iron; C022616:thiazolyl blue