| 摘要 |
<p>Titration is an important and critical step in dosing recombinant virus for gene therapy. A relatively fast, convenient and sensitive method that allows for precise quantification of recombinant retrovirus is presented. 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 -labeled nucleotides as a substrate for chain elongation, the neo-synthetic DNA is labeled by an FITC-conjugated anti- antibody. To avoid the possibility of false positives, the puromycin resistance gene, which is associated with the transgene in the same viral vector and is not normally present in mammalian cells was amplified. The retroviral titer was evaluated by counting FITC-positive cells after PRINS labeling, while knowing the number of cells that were transduced with different amounts of viral supernatant. A comparable viral concentration of 1 X 107 infectious units/mL was found among the retroviruses.</p> |
