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)...
| Main Authors: | , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
2009
|
| _version_ | 1797067109019156480 |
|---|---|
| author | El-Sherbini, Y Stevenson, M Seymour, L Wade-Martins, R |
| author_facet | El-Sherbini, Y Stevenson, M Seymour, L Wade-Martins, R |
| author_sort | El-Sherbini, Y |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T21:51:36Z |
| format | Journal article |
| id | oxford-uuid:4b79b165-2764-4012-baed-150b0f3884cb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:51:36Z |
| publishDate | 2009 |
| record_format | dspace |
| spelling | oxford-uuid:4b79b165-2764-4012-baed-150b0f3884cb2022-03-26T15:43:46ZQuantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:4b79b165-2764-4012-baed-150b0f3884cbEnglishSymplectic Elements at Oxford2009El-Sherbini, YStevenson, MSeymour, LWade-Martins, RHerpes 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. |
| spellingShingle | El-Sherbini, Y Stevenson, M Seymour, L Wade-Martins, R Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR. |
| title | Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR. |
| title_full | Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR. |
| title_fullStr | Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR. |
| title_full_unstemmed | Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR. |
| title_short | Quantitative characterization of cell transduction by HSV-1 amplicons using flow cytometry and real-time PCR. |
| title_sort | quantitative characterization of cell transduction by hsv 1 amplicons using flow cytometry and real time pcr |
| work_keys_str_mv | AT elsherbiniy quantitativecharacterizationofcelltransductionbyhsv1ampliconsusingflowcytometryandrealtimepcr AT stevensonm quantitativecharacterizationofcelltransductionbyhsv1ampliconsusingflowcytometryandrealtimepcr AT seymourl quantitativecharacterizationofcelltransductionbyhsv1ampliconsusingflowcytometryandrealtimepcr AT wademartinsr quantitativecharacterizationofcelltransductionbyhsv1ampliconsusingflowcytometryandrealtimepcr |