[Construction, packaging and titration of recombinant adeno-associated virus vectors contaning antitumor genes].
OBJECTIVE
To construct, package, and titrate vectors that express antitumor genes using adeno-associated virus (AAV) containing human alpha-fetoprotein (AFP) promoter.


METHODS
Recombinant AAV vectors were constructed by blunted ligation of plasmids rAAV-AFP-GFP and pTR-UF5 and p16 cDNA and p21 cDNA. Packaging cell lines, 293 and C12 cells, were transfected with rAAV vectors, plasmid PspRC72, and adenovirus cosmid pAdc with the help of LipofectAMINE and then infected by wild adenovirus 48 hours later. The total viral particle titre was measured by PCR. Electron microscopy was used to observe the morphology of rAAV. C12 cells were infected with rAAV and wild adenovirus (MOI 10) and the titre of infectious virus was measured. by RCA method. Hepatocellular carcinoma cells were cultured and infected with rAAV containing green fluorescent protein (GFP), the infectious rate was observed 48 hours later.


RESULTS
Recombinant vectors, rAAV-AFP-p16, rAAV-AFP-p21, rAAV-CMV-p16, and rAAV-CMV-p21, were constructed and verified by DNA sequencing and enzyme digestion. The total viral titre was 10(13) to 10(14)/ml. The titre of infectious rAAV containing GFP was 5 x 10(10)/ml. Electron microscopy showed that the rAVV was round and plaqued with a diameter between 20 and 30 nm. When the MOI was 100, the infectious rate of hepatocellular carcinoma cells was at least 70%. Four rAAV vectors carrying p16 or p21 genes have been constructed. rAAV packaging and titration methods were developed to produce highly-purified AAV stocks. These approaches help in research of gene therapy in liver cancer.
Zhang
Youbin
Y
Department of Hepatobiliary Surgery, People's Hospital, Peking University, Beijing 100044, China.
Leng
Xisheng
X
Peng
Jirun
J
He
Xiangjun
X
Lu
Jianfeng
J
Weng
Shangeng
S
Wang
Shenwu
S
Du
Jiayu
J
D002460:Cell Line; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D000229:Dependovirus; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D017186:Titrimetry; D000509:alpha-Fetoproteins / Q000235:genetics
C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008164:Luminescent Proteins; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins