kat: a high-efficiency retroviral transduction system for primary human T lymphocytes

Finer, MH; Dull, TJ; Qin, Lu; Farson, Deborah; Roberts, MR

doi:10.1182/blood.v83.1.43.bloodjournal83143

Cited by 48 publications

(55 citation statements)

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“…The rKat retroviral vector system was used as previously described (6). Retroviral supernatant was produced from transiently transfected 293T cells using a modified calcium chloride transfection protocol that has been previously described (5).…”

Section: Methodsmentioning

confidence: 99%

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

et al. 2009

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Co-immunoprecipitation is the classical approach for investigating protein-protein interactions. Analysis is generally conducted using the Western blot approach. We set out to investigate whether flow cytometry was a feasible alternative to Western blotting. Using the TCR-CD3 complex as a model for intermolecular interactions in the MA5.8 cell line, FLAG-tagged CD3f-scFv fusion proteins could be captured on anti-FLAG coupled beads and associated TCRb molecules could be detected by flow cytometry. This association was abrogated by mutations to the CD3f transmembrane domain. Using multicolor flow cytometry, TCRb, CD3e, and the scFv region of the CD3f fusion molecule could all be detected from a single sample. This multicolor analysis was then applied to demonstrate the importance of correct lysis conditions for extraction of the TCR complex. In summary, this flow cytometric immunoprecipitation technique is a feasible alternative to classical co-immunoprecipitation analysis technique and offers many potential advantages including rapid analysis with increased target sensitivity, reduced technical demands, amenable to multiple protein analysis from a single sample, and provides a framework that may facilitate the development of high throughput analytical assays investigating protein-protein interactions. ' 2009 International Society for Advancement of Cytometry Key termsT-cell receptor; CD3f; co-immunoprecipitation; protein-protein interactions THE co-immunoprecipitation/Western blotting (co-ip/WB) approach has been the gold-standard technique for the investigation of suspected protein-protein interactions for many years. However, the co-ip/WB approach has limitations. For example, heavy and light chains from the capture antibodies can obscure identification of immuno-reactive bands on immuno-blots of the same molecular weight as the protein of interest. Furthermore, SDS-PAGE/WB can be a time-consuming process, particularly if multiple protein interactions are being investigated. To circumvent some of the issues surrounding standard co-ip/WB, we investigated the use of flow cytometry to analyze immunoprecipitates from a well-documented protein complex. The advantage of this approach being that the high throughput approach of flow cytometry would expedite the identification process and, with the large number of fluorochrome conjugated antibodies now available to the researcher, would permit analysis of multiple protein-protein interactions. Bead-based assays have steadily become adapted for flow cytometric purposes and can now be used to analyze kinase inhibitors (1), to quantify antibody binding sites (2), to detect low concentrations of DNA (3), and to measure cytokine secretion (4). Here, we show that a bead-based flow cytometric immunoprecipitation assay (FCIP) can be used to successfully analyze interactions between CD3f and the T-cell receptor (TCR) complex. Not only is

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Section: Methodsmentioning

confidence: 99%

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

et al. 2009

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“…Rat thyroid PC C13 cell lines immortalized with either E1A or v-Raf have been described previously (1). Primary human T lymphocytes were isolated and transduced as previously described (8). Gamma irradiation (8,000 rads) was delivered to cells in suspension as in previous studies (31) by a 3-min exposure to a 60 Co source.…”

Section: Sin Plasmids (I) Phrsin Plasmidsmentioning

confidence: 99%

Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery

Zufferey

Dull

Mandel

et al. 1998

J Virol

1,586

530

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In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3′ long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.

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“…The limitations associated with murine packaging cells have led some researchers to design packaging cells derived from human cells. The human embryonic kidney cell line, 293, has been shown to produce virus that yields higher transduction efficiencies (22, 68), as compared to virus obtained from Ψ‐CRIP‐based producer cells (17). Because packaging cell lines derived from human cells lack endogenous murine retroviral sequences, the likelihood of producing RCRs is minimized (17).…”

Section: Large‐scale Production and Downstream Processingmentioning

confidence: 99%

Large-Scale Processing of Recombinant Retroviruses for Gene Therapy

et al. 1999

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Gene therapy is a new therapeutic modality with the potential of treating inherited and acquired diseases. Several viral and physicochemical vehicles have been used for the transfer of genes to mammalian cells, but recombinant retroviruses are used in the majority of gene therapy clinical trials today. In this communication, we review the major concerns associated with the large-scale production and processing of retroviral particles. While some of the current processes for manufacturing recombinant proteins will be applicable to recombinant retroviruses, the instability, sensitivity to inhibitors, complexity, and size of retroviral particles require that new technologies be designed and evaluated. Here, we examine those issues critical to the design of strategies for production, concentration, and purification as well as formulation and storage of recombinant retroviruses. Processes for large-scale manufacturing of recombinant retroviruses that can produce high gene transfer efficiencies will have significant impact on the clinical implementation of gene therapy.

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kat: a high-efficiency retroviral transduction system for primary human T lymphocytes

Cited by 48 publications

References 0 publications

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery

Large-Scale Processing of Recombinant Retroviruses for Gene Therapy

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