Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency.
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 Moloney murine 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-consuming tissue 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 or both qPCR targets.
Mackay
I M
IM
Gene Therapy Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston Road, Herston, Queensland 4029, Australia. i.mackay@mailbox.uq.edu.au
Metharom
P
P
Sloots
T P
TP
Wei
M Q
MQ
D000818:Animals; D004587:Electrophoresis, Agar Gel; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D019868:Kanamycin Kinase / Q000096:biosynthesis; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase