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 Tag 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.
Application of the primer in situ DNA synthesis (PRINS) technique to titer recombinant virus and evaluation of the efficiency of viral transduction
Cinti C Cinti C;
2001
Abstract
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 Tag 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
