[Construction of adeno-associated virus vector carried mutated dihydrofolate reductase and green fluorescent protein and its expression in NIH3T3 cells].
The aim of this study was to construct recombinant mDHFR-GFP/AAV vector containing mutated dihydrofolate reductase (mDHFR) and green fluorescent protein (GFP) fusion genes and its expression in NIH3T3 cells, to investigate the resistance of the cells to methotrexate. Amplified cDNA of mDHFR and GFP segmented from their plasmid separately were linked by PCR with the aminoacetic acid linker. The fusion gene was inserted into T vector, and after enzyme cutting the fusion gene fragment was inserted into AAV vector, then packaging the vector into recombined AAV and infected NIH3T3 cells. Expression of gene fusion was observed by PCR, fluorescent microscopy and flow cytometry. mDHFR and GFP cDNA were found in NIH3T3 genomic DNA, the GFP expression rate was about 25%, and resistance of the transferred cells to MTX was increased markedly. The results showed that AAV vector can transfer mDHFR and GFP fusion gene into NIH3T3 cells and increase resistance to MTX in gene modified cells. This data provided a basis for application of mDHFR and AAV vector in gene therapy.
Li
Li-Bo
LB
Department of Oncology, Nanfang Hospital, The First Military Medical University, Guangzhou 510515, China.
Feng
Ru
R
Zhou
Shu-Yun
SY
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics*; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000818:Animals; D002470:Cell Survival; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; D004262:DNA Restriction Enzymes