Chemical methods for transfection include the use of cationic liposomes (lipoplex), polymers (polyplex), combinations of the two (lipopolyplex), calcium phosphate, and DEAE dextran. In addition to the chemical methods, a number of physical methods exist that pro- mote the direct entry of uncomplexed DNA into the cell. These methods can include microinjection of individual cells, hydroporation, electroporation, ultrasound, and biolistic delivery.
2 μl of Lipofectamine2000 and 2 μg of pEGFP-N1 were diluted separately in 50 μl of Opti-MEM reduced serum medium and mixed gently. After 5 min incubation at room temperature, the Lipofect-amine2000 and pEGFP-N1 were combined and incubated for an additional 25 min at room temperature to allow the DNA-Lipofect-amine2000 complexes to form. The complexes were added to cells grown in Opti-MEM medium without serum and antibiotic. After 6 hr the medium was replaced with RPMI-1640 supplemented with 10% fetal bovine serum.
4 μl of jetPEI and 2 μg of pEGFP-N1 were diluted with 50 μl of 150 mM NaCl. The DNA solution was then added to the jetPEI solution, and after 20 min incubation at room temperature, 100 μl of the complexes were added to cells grown in serum containing medium.
2 μg of pEGFP-N1 was mixed with 100 μl of LyoVec and incubated at room temperature for 15 – 30 min to allow the formation of the complex. Subsequently 25 μl of the complex was added to cells grown in serum containing RPMI-1640 medium.
Efficiency of 71.2%, 57% and 22.2% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine2000, jetPEI and LyoVec reagents, respectively
SP2/0, NS1 and P3U1 myeloma cell lines were hardly transfected by transfection reagents. NS0 and Ag8, however, were efficiently transfected by Lipofectamine 2000 and jetPEI reagents.
Immunization of mice with transfected murine myeloma cell lines is considered as an excellent alternative. In several studies SP2/0 myeloma cells transfected with different genes were used to express recombinant pro- teins, however, no data was reported regard- ing the transfection efficiency of the target DNA in this cell line.
Lipoplexes are internalized by cells solely by means of clathrin-mediated endocytosis, while poly- plexes which are composed mainly of inorganic polymers, such as polyethyleneimine, are internalized both by clathrin-mediated and by caveolae mediated endocytosis.
Defects in endocytosis in myeloma cell lines may contribute to their low transfection efficiency. Some studies have demonstrated differential gene expression levels induced by special promoters in different cells because transgene expression by plasmid vectors benefits from the use of cellular transcriptional regulatory elements that permit high- level gene expression