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Titolo:
Application of the polymerase chain reaction in determination of recombinant retrovirus titers as fifty percent endpoints
Autore:
Husemoen, LLN; Gram, GJ; Hansen, JES;
Indirizzi:
Univ Copenhagen, Hvidovre Hosp, Infect Dis Lab 144, DK-2650 Hvidovre, Denmark Univ Copenhagen Hvidovre Denmark DK-2650 144, DK-2650 Hvidovre, Denmark
Titolo Testata:
APMIS
fascicolo: 1, volume: 108, anno: 2000,
pagine: 38 - 44
SICI:
0903-4641(200001)108:1<38:AOTPCR>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
FLUORESCENT PROTEIN GENE; LEUKEMIA-VIRUS; CELL-LINE; VECTORS; EXPRESSION; ASSAY; LYMPHOCYTES; SYSTEM;
Keywords:
retroviral vectors; virus titer; quantification; RT-PCR; PCR; Reed-Muench titration;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
30
Recensione:
Indirizzi per estratti:
Indirizzo: Gram, GJ Univ Copenhagen, Hvidovre Hosp, Infect Dis Lab 144, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark Univ Copenhagen Kettegaard Alle 30 Hvidovre Denmark DK-2650 mark
Citazione:
L.L.N. Husemoen et al., "Application of the polymerase chain reaction in determination of recombinant retrovirus titers as fifty percent endpoints", APMIS, 108(1), 2000, pp. 38-44

Abstract

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. One of the most laborious routine assays in the application of retroviral-mediated gene transfer is the determination of viral titers of vector producer cell lines. Traditionally, determination of virus titer involves the testing of culture medium from individual packaging cell lines for the ability to transfer drug resistance to susceptible cells - a process that can easily take up to 14 days. It is generally agreed that this method is cumbersome. We sought to develop PCR-based protocols that would significantly simplify and shorten this procedure. Using PCR and primers specific for the Neo-region of the MLV-derived vector LeGSN, we determined 1. the proviral integration in target cells, and 2. the viral nucleic acid (RNA or DNA) content of the vector stock. Results werecompared with those using the conventional method. We found that these specific PCR-based procedures were indeed useful for rapid determination of viral titers as well as for quick screening for high-titer vector-producing cell crones and successful transduction of target cells.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 24/11/20 alle ore 14:13:24