A method to estimate the efficiency of gene expression from an integrated retroviral vector.
BACKGROUND
Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of proviral gene expression while vigorously excluding the contribution from unstable species such as passively transferred plasmid and LTR circles. Here, we present a method that can achieve this.


RESULTS
Proviral gene expression was detected by the activity of the puromycin resistance gene encoded in the viral vector, and quantified by comparing the growth curve of the sample under puromycin selection to that of a series of calibration cultures. Reproducible estimates of the efficiency of proviral gene expression could be derived. We confirm that contamination from unstable species such as passively transferred plasmid used in viral vector production and unintegrated viral DNA can seriously confound estimates of the efficiency of transduction. This can be overcome using a PCR based on limiting dilution analysis.


CONCLUSIONS
A simple, low cost method was developed that should be useful in studying the biology of retroviruses and for the development of expression systems for retrovirus based gene therapy.
Mok
Hoi Ping
HP
Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hill's Road, Cambridge, CB2 2QQ, UK. hpm22@cam.ac.uk
Lever
Andrew
A
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011533:Proviruses / Q000235:genetics / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection; D014773:Virology / Q000379:methods; D016662:Virus Integration
D004279:DNA, Viral; D011500:Protein Synthesis Inhibitors; D011691:Puromycin