Application of the primer in situ DNA synthesis (PRINS) technique to titer recombinant virus and evaluation of the efficiency of viral transduction.
Titration is an important and critical step in dosing recombinant virus for gene therapy. We present a relatively fast, convenient, and sensitive method that allows for precise quantification of recombinant retrovirus. The method is based on PCR amplification of a foreign gene by the PRINS (primer in situ DNA synthesis) technique. The PRINS technique is based on the sequence-specific annealing of unlabeled oligonucleotide DNA in situ. This oligonucleotide operates as a primer for in situ chain elongation catalyzed by the Taq I polymerase. Using digoxygenin-labeled nucleotides as a substrate for chain elongation, the neo-synthetic DNA is labeled by an FITC-conjugated anti-digoxygenin antibody. To avoid the possibility of false positives, we amplified the puromycin-resistance gene, which is associated with the transgene in the same viral vector and is not normally present in mammalian cells. The retroviral titer was evaluated by counting fluorescein isothiocyanate-positive cells after PRINS labeling, while knowing the number of plated cells that were transduced with different amounts of viral supernatant. A comparable viral concentration of 1 x 10(7) infectious units/mL was found among the retroviruses.
Claudio
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Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, PA 19107, USA. pclaudio@lac.jci.tju.edu
Cinti
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Giordano
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D000230:Adenocarcinoma / Q000235:genetics / Q000628:therapy; D000818:Animals; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000628:therapy; D017931:DNA Primers / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D017403:In Situ Hybridization; D007680:Kidney Neoplasms / Q000235:genetics / Q000628:therapy; D008175:Lung Neoplasms / Q000235:genetics / Q000628:therapy; D051379:Mice; D008819:Mice, Nude; D016133:Polymerase Chain Reaction / Q000379:methods*; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D005456:Fluorescent Dyes