Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.
The use of lentiviral vectors for basic research and potential future clinical applications requires methodologies that can accurately determine lentiviral titers and monitor viral transgene expression within target cells, beyond the context of reporter genes typically used for this purpose. Here we describe a quantitative RT-PCR (qRT-PCR) method that achieves both goals using primer sequences that are specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an enhancer contained in many retroviral vectors and that is incorporated in the 3' UTR of nascent transgene transcripts. Quantitation of titers of three recombinant lentiviruses, genetically identical except for the transgene, demonstrated consistent differences in titer that were likely due to transgene-associated toxicity in producer cells and target cells. Viruses encoding the tumor-associated antigens tyrosinase and neo-poly(A) polymerase yielded reproducibly lower titers than a virus encoding enhanced green fluorescent protein (GFP) at the viral RNA, integrated DNA, and transgene mRNA levels, as measured by WPRE qPCR. Furthermore, the magnitude of differences in expression of the three transgenes in transduced target cells could not have been predicted by measuring vector DNA integration events. Since transgene expression in target cells is the most common goal of lentiviral transduction, and since methods to quantify transgene expression on the protein level are not always readily available, qRT-PCR based on a nucleotide sequence included in the transcript provides a useful tool for titering novel recombinant lentiviruses.
Lizée
Gregory
G
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Aerts
Joeri L
JL
Gonzales
Monica I
MI
Chinnasamy
Nachimuthu
N
Morgan
Richard A
RA
Topalian
Suzanne L
SL
D020413:3' Untranslated Regions / Q000235:genetics; D002460:Cell Line; D002461:Cell Line, Transformed; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005809:Genes, Regulator / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006518:Hepatitis Viruses / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014161:Transduction, Genetic; D019076:Transgenes; D014771:Virion / Q000235:genetics; D016662:Virus Integration
D020413:3' Untranslated Regions; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins