Phagocytosis-mediated retroviral transduction: co-internalization of deactivated retrovirus and calcium-alginate microspheres by macrophages.
BACKGROUND
Efficiency of retrovirus-mediated gene transfer has been hampered by short retroviral half-life due to shedding of retroviral envelope proteins which is of utmost importance to the interaction between retrovirus and cell.


METHODS
Calcium-alginate microspheres with average size of 780 nm were prepared by reverse emulsification and characterized by scanning electron microscopy and microelectrophoresis. To obtain deactivated retroviruses produced from 293 packaging cells, retrovirus-containing media were pre-incubated at 37 degrees C for 6, 12, 18, and 24 h, respectively. Murine J774A.1 macrophages were co-treated with Ca-alginate microspheres and deactivated retroviruses encoding the enhanced green fluorescent protein (eGFP) gene. Through phagocytosis, deactivated retroviruses and Ca-alginate microspheres were co-internalized into macrophages. After retroviral transduction for 24 and 48 h, the percentages of macrophages with eGFP expression were determined by bright-field and fluorescence microscopy.


RESULTS
After 48-h incubation with Ca-alginate microspheres and deactivated retroviruses, phagosomes turned into large vacuoles occupied almost half of the cytoplasmic space. This was probably attributed to the erosion of Ca-alginate microspheres by destructive agents within vacuolar compartments and concomitant osmotic swelling. It was reasoned that deactivated retroviruses escaped such enlarged vesicles easily and underwent reverse transcription in the cytosol. The expression of eGFP in macrophages infected by retroviruses pre-incubated for 24 h in the presence of Ca-alginate microspheres was thereby augmented up to tenfold in comparison with the cells treated with 24-h deactivated retroviruses only.


CONCLUSIONS
Ca-alginate microspheres performed as auxiliary agents for the enhancement of retrovirus-mediated gene transfer efficiency even though retroviruses had been deactivated due to the loss of envelope proteins.
You
Jin-Oh
JO
Department of Chemical Engineering, University of Southern California, Los Angeles, CA 90089-1211, USA.
Peng
Ching-An
CA
D000464:Alginates; D000818:Animals; D002460:Cell Line; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008863:Microspheres; D010587:Phagocytosis; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid