Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR.
Herpes simplex virus type 1 (HSV-1) amplicon preparations are usually quantified as transducing units/ml (TU/ml), with little information on genomic copy/TU ratios. In the present study, two HSV-1 amplicons expressing enhanced green fluorescent protein (EGFP) were analysed by quantitative PCR (qPCR) and transducing activity to obtain genomic copy/TU ratios. One vector (pHSV-GL) contains the HSV-1 packaging signal (pac) and origin of replication (oriS) and the other (pHSV/EBV-GL) includes Epstein-Barr virus (EBV) episomal maintenance elements. The pHSV-GL and pHSV/EBV-GL amplicons were prepared at titres of 7.55x10(7) and 7.24x10(7)TU/ml, containing 2.56x10(9) and 1.33x10(9) genomic copies/ml respectively. This produced preliminary estimates of genomic copy/TU ratios of 34:1 and 18:1. However standard transduction conditions did not deplete fully the supernatant of transducing particles since the same supernatant was subsequently able to achieve 25% the initial transduction efficiency, although centrifugation of amplicon particles onto cells improved infectivity by 1.8-fold. Finally, qPCR analysis of FACS-purified EGFP-expressing cells showed the presence of approximately 3 amplicon genomes/transduced cell, independent of the infection dose. Accordingly, the initial estimated genomic copy/TU ratio for pHSV-GL was revised to 6.3:1. Measuring the genomic copy/TU ratios is an important parameter for comparing the quality of amplicon preparations and standardizing experimental conditions.
El-Sherbini
Yasser M
YM
Department of Clinical Pharmacology, Old Road Campus Research Building, University of Oxford, Oxford OX3 7DQ, UK.
Stevenson
Mark M
MM
Seymour
Leonard W
LW
Wade-Martins
Richard
R
D000818:Animals; D002522:Chlorocebus aethiops; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D014161:Transduction, Genetic; D014709:Vero Cells
D049452:Green Fluorescent Proteins