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Titolo:
Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency
Autore:
Mackay, IM; Metharom, P; Sloots, TP; Wei, MQ;
Indirizzi:
Royal Childrens Hosp, Sir Albert Sakzewski Virus Res Ctr, Clin Virol Res Unit, Herston, Qld 4029, Australia Royal Childrens Hosp Herston Qld Australia 4029 ston, Qld 4029, Australia Royal Childrens Hosp, Sir Albert Sakzewski Virus Res Ctr, Gene Therapy Unit, Herston, Qld 4029, Australia Royal Childrens Hosp Herston Qld Australia4029 ston, Qld 4029, Australia Univ Queensland, Dept Microbiol, Brisbane, Qld 4072, Australia Univ Queensland Brisbane Qld Australia 4072 Brisbane, Qld 4072, Australia Univ Queensland, Dept Pediat & Child Hlth, Brisbane, Qld 4072, Australia Univ Queensland Brisbane Qld Australia 4072 Brisbane, Qld 4072, Australia Univ Queensland, Clin Med Virol Ctr, Brisbane, Qld 4072, Australia Univ Queensland Brisbane Qld Australia 4072 Brisbane, Qld 4072, Australia
Titolo Testata:
MOLECULAR THERAPY
fascicolo: 5, volume: 3, anno: 2001,
parte:, 1
pagine: 801 - 808
SICI:
1525-0016(200105)3:5<801:QPFTDO>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
POLYMERASE-CHAIN-REACTION; RT-PCR; GENE-TRANSFER; LENTIVIRUS VECTOR; MOLECULAR METHODS; DETECTION SYSTEM; INTERNAL CONTROL; IN-VIVO; VIRUS; ASSAY;
Keywords:
quantitation; PCR-ELISA; transduction; retroviral vector; gene therapy; MoMLV;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Mackay, IM Royal Childrens Hosp, Sir Albert Sakzewski Virus Res Ctr, Clin Virol Res Unit, Herston Rd, Herston, Qld 4029, Australia Royal Childrens Hosp Herston Rd Herston Qld Australia 4029 alia
Citazione:
I.M. Mackay et al., "Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency", MOL THER, 3(5), 2001, pp. 801-808

Abstract

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloneymurine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consumingtissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one orboth qPCR targets.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 31/03/20 alle ore 15:30:24