[Production and purification of helper free recombinant adeno-associated virus 2].
OBJECTIVE
To explore an efficient and simple way to product and purify helper free adeno-associated virus vectors.


METHODS
Helper free rAAV system was transferred into HEK293T cells through phosphorated calcium method, thus producing rAAV, then the rAAV vector was purified through chloroform-PEG8000/NaCl-chloroform method and ultrafiltration. SDS-PAGE protein electrophoresis and Western blotting were used to detect the rAAV protein. The quantity of rAAV genome was determined through blot hybridization. HEK293T cells were cultured, rAAV was added, and fluorescence microscopy was used to count the amount of cells expressing green fluorescent protein so as to measure the transferring unit of rAAV.


RESULTS
rAAV2 was thus produced with a particle number of 2 x 10(13)/ml and a transferring unit of 5 x 10(11)/ml.


CONCLUSIONS
This method to produce and purify rAAV is efficient and simple, without need of any special equipment, and can be finished in a common laboratory.
Li
Gui-lin
GL
Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing 100730, China.
Wang
Ren-zhi
RZ
Wang
Xin
X
Dou
Wan-chen
WC
Zhang
Bo
B
Tian
Shi-qiang
SQ
Ren
Zu-yuan
ZY
Su
Chang-bao
CB
D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014162:Transfection; D019065:Virus Assembly; D014779:Virus Replication
D017931:DNA Primers