Construction and replication of an adeno-associated virus expression vector that contains human beta-globin cDNA.
With the goal of using adeno-associated viruses (AAV) as the gene-transfer vector for gene therapy of hemoglobinopathies, human beta-globin cDNA was ligated downstream from the P40 promoter of the AAV type-2 (AAV2) genome. To circumvent difficulties of cloning DNA containing palindromic sequences, two of which exist in the termini of AAV genome, a step-wise approach handling one palindrome at a time was devised to construct the chimeric expression vector. Electroporation of the construct into human 293 cells (embryonal kidney cell line) resulted in expression of the cloned human beta-globin cDNA, as evidenced by the synthesis of transcripts hybridizable to human beta-globin cDNA probe. Addition of the 3'-end region of AAV DNA that contains both the transcription termination signal and origin of DNA replication for AAV to the construct permitted the recombinant AAV genome to be rescued and replicate in the cell.
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Department of Biochemistry, School of Medicine, Meharry Medical College, Nashville, TN 37208.
Dixit
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Tillery
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Plonk
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D004247:DNA / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
D004279:DNA, Viral; D005914:Globins; D004247:DNA