Magnetofection and Ultrasonication

Topics: Cell, DNA, Gene Pages: 6 (1731 words) Published: April 23, 2013
Gene transfer is the introduction of foreign genetic material either DNA or RNA, artificially or naturally into a cell. The true benefits of gene therapy cannot be realized until current gene delivery systems are perfected or new vectors are developed. According to Plank .C (2003); the main problems currently associated with systemic gene vector administration (gene therapy) include bio-distribution of gene vector throughout the body, lack of specificity towards a pathological site (bioavailability at the target site), necessity of a large dose to achieve high local concentration, non-specific toxicity, slow vector accumulation and consequently low vector concentration at target tissues have been identified as simple but strong barriers to effective gene delivery. Magnetofection is defined as nucleic acid delivery under the influence of magnetic field acting on nucleic acid vectors that are associated with magnetic nanoparticles. It is a direct method of gene transfer. It is a highly efficient and simple method of transfecting cells in culture (in-vitro) and in vivo. Schwelrdt et al 2012 says; it is based on the concept of magnetic drug targeting of gene vectors and it can be applied in gene delivery with viral and non-viral vectors. Magnetic targeting exploits paramagnetic particles as drug carriers, guides their accumulation in target tissues with local strong magnetic fields. The principle is to associate magnetic nanoparticles with nucleic acids, viruses and transfection reagents and target gene delivery by application of a magnetic field. This technique of gene transfer was developed mainly to solve the problem related to diffusion limited process for in vitro applications and to restricted bioavailability at the target site for in vivo applications. Magnetofection works for primary cells and hard to transfect cells that are not dividing or slowly dividing, meaning that the genetic materials can go to the cell nucleus without cell division. Coupling magnetic nanoparticles to gene vectors of any kind; results in a dramatic increase of the uptake of these vectors and consequently high transfection efficiency (Sapet et al; 2010). Magnetofection technology requires transfection reagents. In industry two ready to use transfection reagents are available these are PolyMAG and CombiMAG; they are composed of magnetic nanoparticles and associated with a proprietary polymer. PolyMAG is a cationic polymer based magnetic formulation. It is mixed with the nucleic acid to be transfected. CombiMAG together with Dogctor form a cationic lipid based magnetic formulation. This reagent is suitable for plasmid DNA, antisense oligonucleotides, mRNA, shRNA and siRNA. It also requires appropriate magnetic fields that magnetizes nanoparticles in solution, forms a very strong gradient to attract the nanoparticles and covers all the surface of the plate. To perform efficient transfection or infection, suitable magnetic nanoparticles formulations and magnetic field, are the only necessity. Therefore, two optimized magnetic plates with improved properties have been especially designed for magnetofection. Their special geometry and organization produce a strong magnetic field that is suitable for all cell culture dishes and supports (Knots F et al; 2003). Mechanism

It involves the vortex of PolyMAG before use. The required amount of magnetic particles (PolyMAG) is added in a microcentrifuge tube in accordance to the gene vector (DNA, virus and siRNA) amount to be used. The required amount of DNA to be transfected is added which has been diluted with serum-supplement free medium (DMEM). Vigorous pipetting is done immediately to mix the contents. Vector association with transfecting reagent is mediated by electrostatic interaction and salt-induced colloid aggregation. After 15minutes incubation, the magnetic particles/DNA complexes are added to cells complexes. The 96 well cell culture plate is placed on the Magnetofactor plate for up to 10 – 20...
Continue Reading

Please join StudyMode to read the full document

Become a StudyMode Member

Sign Up - It's Free