[Sorting of packaging cells for retroviral vector carrying green fluorescent gene and viral titer determination].
OBJECTIVE
To develop a method for acquisition of efficient and stable retroviral packaging cells on the basis of fluorescence-activated cell sorting (FACS) technique.


METHODS
PA317 cells were transfected by the recombinant retroviral vector pLEGFP via liposome, and the result of transfection was examined using fluorescent microscope. Polyclones and monoclone of the packaging cells were obtained with FACS and identified by PCR and reverse transcriptional PCR (RT-PCR). NIH3T3 cells were used to determine the virus titer in the supernatant.


RESULTS
Examination under fluorescence microscope confirmed the success of cell transfection with the retrovirus, and polyclonal and monoclonal cells with efficient and stable expression were obtained after FACS. The inserted EGFP gene fragment could be amplified by PCR from the genomic DNA of the polyclonal and monoclonal cells and by RT-PCR from the retrovirus RNA in the supernatant of monoclonal cell culture and some of the monoclonal cell cultures (6/8). Determination of the virus titer in the cell culture supernatant showed efficient viral production by the cells.


CONCLUSIONS
FACS is an efficient method for obtaining stable retroviral packaging cells.
Zhang
Yin-gang
YG
Institute of Endemic Disease, Education Ministry Key Laboratory of Environment and Disease-Related Genes, Xi'an Jiaotong University, Xi'an 710061, China. zyingang@263.net
Guo
Xiong
X
Zhou
Jing-jun
JJ
Yu
Bing
B
Liu
Bing
B
D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D010948:Viral Plaque Assay / Q000379:methods
D049452:Green Fluorescent Proteins