Rapid titer determination using quantitative real-time PCR

Abstract

Quantitative real-time PCR was utilized to evaluate retrovilated with biologic titers performed on the test material. The ral vector titer. RNA was prepared from vector supernatant 96-well capacity of the machine and 2 h of running time and run in a one-step RT-PCR reaction combining reverse permit titer determinations within 8 h, facilitating the protranscription (RT) and amplification in one tube. Sample cessing of large sample numbers while greatly decreasing analysis was performed in the ABI Prism 7700 Sequence technician time. Real-time PCR improves titer quantifiDetector. PCR was quantitative over a range of 10 1 to cation and the identification of high-titer producer cells. 6 × 10 5 vector particles per reaction (2 × 10 2 to 1 × 10 7 vecThis methodology will help investigators meet the chaltor particles per millilites of supernatant) and closely correlenges of developing vectors which lack selectable markers.Keywords: retroviral vectors; vector production; vector titer; quantitative PCR Estimation of retroviral vector titer and identification of high-titer packaging cell lines have been aided by the incorporation of selectable markers within the vector genome. These markers have typically been drug selection genes 1-4 or molecules expressed on the cell surface. 5 Recent concern about the immunogenic potential of these gene products in animals models and human clinical trials have prompted investigators to remove drug selection genes from vector constructs. 6,7 To simplify titer determination, and facilitate rapid identification of hightiter producer clones, investigators have utilized PCR and RNA dot blot assays to estimate vector RNA in supernatant preparations. [8][9][10] These methods are helpful in separating vectors with large to modest differences in titer but remain semi-quantitative. The development of real-time PCR technology, which combines PCR and fluorescence detection of target sequences, may provide a method for quantitative PCR with increased accuracy over current methods. 11,12 In addition, this technology provides for processing of large number of samples (96 reactions per run), one-step RT-PCR reactions (reverse transcription and PCR amplification in one tube), and real-time read-out (without the need for electrophoresis or dot blot analysis). This article demonstrates how this methodology accurately predicts biologic titers, significantly decreases turn around time, and decreases technician time. It has greatly simplified the screening of vector producer cells for high titer clones. Real-time PCR was performed using the ABI Prism 7700 Sequence Detector (TaqMan; PE Applied Biosystems, Foster City, CA, USA) which combines PCR tech-

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

“…Nevertheless, the RNA method could be used for comparative analysis of production supernatants when screening for material with moderate to high titers. 4,5 In conclusion, of the three methods tested, titers assessed by DNA analysis of transduced cells provide a reliable estimate of functional titers as they account for factors such as defective interfering particles, vector expression levels, and plasmid contamination. The realtime PCR method described here can also be used for functional titer determination of a wide variety of vectors irrespective of transgenes.…”

“…[4][5][6]9 However, the RNA titers thus obtained may overestimate the number of functional vector particles due to the presence of defective interfering particles and packaging cell line DNA. 10,17,20 Also, as shown by our studies above with the amp r PCR assay, carry-over of plasmid DNA is of concern in vectors produced by transient transfection methods.…”

“…Currently a variety of methods for assessing titer are available, but a comprehensive study of lentiviral titer methodology has not been performed. [4][5][6][7][8][9][10][11] Lentiviral vectors possess many characteristics similar to murine-based retroviral vectors, but their production methods often differ significantly (transient transfection versus packaging cell lines, respectively). 12 3 …”

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years

“…From the start of our experiments in 1998 we have chosen to assess the transduction potency of the vector supernatants on primary human T cells using optimized clinical-scale protocols. We choose transduction efficiency to be a surrogate marker for vector stability for the following reasons: (1) our retroviral vector lacks a selection marker, which would facilitated other/nonfunctional quantification methods; [18][19][20] (2) PCR-based methods would have been unable to discriminate defective virus particles or detect the presence of inhibitors of transduction 21,22 and (3) infection of compatible cell lines such as 293 and Mus Dunni for GaLV-and MLV-pseudotyped vectors, respectively, demonstrate considerable biovariability (data not shown).…”

Expansion of tropism of a feline parvovirus to target a human tumor cell line by display of an αv integrin binding peptide on the capsid

“…42 Control experiments involving addition of luciferase plasmid DNA showed that the DNase treatment was effective in eliminating any PCR signal from this source (data not shown). Serially diluted samples were then subjected to PCR using primers for luciferase (5Ј-TGGCCCTTCCGCATAGAA and 5Ј-TGATTGCC AAAAATAGGATCTCTG) together with a quenchedfluorescence-tagged Taqman probe 43 hybridizing to the luciferase sequence between the primers (5Ј-FAM.TGCCTGCGTCAGATTCTCGCATG.TAMRA). PCR was performed (ABI Prism 7700 Sequence Detector, PE Biosystems, Foster City, CA, USA) and quantification was obtained from cycle numbers for threshold appearance of fluorescence, relative to a standard curve for dilutions of linearized luciferase plasmid DNA amplified in parallel (PCR Core facility, UCHSC Cancer Center).…”