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Platinum_GP Retroviral Packaging Cell Line, Pantropic

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[#RV-103] Platinum_GP Retroviral Packaging Cell Line, Pantropic

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RV-103 | Platinum_GP Retroviral Packaging Cell Line, Pantropic, 1 vial
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(1) A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells.[TOP]

Pubmed ID :30394400
Publication Date : //
The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1 or 2 generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Authors : Kueberuwa Gray, Zheng Weiming, Kalaitsidou Milena, Gilham David E, Hawkins Robert E,



(2) Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience.[TOP]

Pubmed ID :30211249
Publication Date : //
Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.

Authors : Cornetta Kenneth, Duffy Lisa, Feldman Steven A, Mackall Crystal L, Davila Marco L, Curran Kevin J, Junghans Richard P, Tang Jean Yuh, Kochenderfer James N, O'Cearbhaill Roisin, Archer Gary, Kiem Hans-Peter, Shah Nirali N, Delbrook Cindy, Kaplan Rosie, Brentjens Renier J, Rivière Isabelle, Sadelain Michel, Rosenberg Steven A,



(3) Lentiviral Vector-Mediated SHC3 Silencing Exacerbates Oxidative Stress Injury in Nigral Dopamine Neurons by Regulating the PI3K-AKT-FoxO Signaling Pathway in Rats with Parkinson's Disease.[TOP]

Pubmed ID :30184529
Publication Date : //
Parkinson's disease (PD) is a prevalent disease that leads to motor and cognitive disabilities, and oxidative stress (OS) injury was found to be related to the etiology of PD. Increasing evidence has shown that SHC3 is aberrantly expressed in neurons. The current study examines the involvement of SHC3 silencing in OS injury in the nigral dopamine neurons in rats with PD via the PI3K-AKT-FoxO signaling pathway.

Authors : Gong Jian, Zhang Lei, Zhang Qian, Li Xiang, Xia Xiang-Jun, Liu Yin-Yuan, Yang Qin-Shang,



(4) Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes.[TOP]

Pubmed ID :30182034
Publication Date : //
Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136-370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

Authors : Tijani Maha, Munis Altar M, Perry Christopher, Sanber Khaled, Ferraresso Marta, Mukhopadhyay Tarit, Themis Michael, Nisoli Ilaria, Mattiuzzo Giada, Collins Mary K, Takeuchi Yasuhiro,



(5) A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion.[TOP]

Pubmed ID :30176017
Publication Date : //
This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

Authors : Esposito Anthony M, Soare Alexandra Y, Patel Foramben, Satija Namita, Chen Benjamin K, Swartz Talia H,



(6) The chromatin binding domain, including the QPQRYG motif, of feline foamy virus Gag is required for viral DNA integration and nuclear accumulation of Gag and the viral genome.[TOP]

Pubmed ID :30145377
Publication Date : //
The retroviral Gag protein, the major component of released particles, plays different roles in particle assembly, maturation or infection of new host cells. Here, we characterize the Gag chromatin binding site including the highly conserved QPQRYG motif of feline foamy virus, a member of the Spumaretrovirinae. Mutagenesis of critical residues in the chromatin binding site/QPQRYG motif almost completely abrogates viral DNA integration and reduces nuclear accumulation of Gag and viral DNA. Genome packaging, reverse transcription, particle release and uptake into new target cells are not affected. The integrity of the QPQRYG motif appears to be important for processes after cytosolic entry, likely influencing incoming virus capsids or disassembly intermediates but not Gag synthesized de novo in progeny virus-producing cells. According to our data, chromatin binding is a shared feature among foamy viruses but further work is needed to understand the mechanisms involved.

Authors : Wei Guochao, Kehl Timo, Bao Qiuying, Benner Axel, Lei Janet, Löchelt Martin,



(7) Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology.[TOP]

Pubmed ID :30002270
Publication Date : //
Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probe the dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) in size, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with a large number of structural and functional intermediates. Single-molecule measurements that reveal the complete distribution of properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessible otherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field of virology. Here, we have focused on new findings in virus-cell interaction, virus cell entry and transport, viral membrane fusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immune proteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges, outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aid our understanding of viruses.

Authors : Banerjee Sunaina, Maurya Satyaghosh, Roy Rahul,



(8) The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag.[TOP]

Pubmed ID :29996845
Publication Date : //
Nuclear localization of Gag is a property shared by many retroviruses and retrotransposons. The importance of this stage for retroviral replication is still unknown, but studies on the Rous Sarcoma virus indicate that Gag might select the viral RNA genome for packaging in the nucleus. In the case of Foamy viruses, genome encapsidation is mediated by Gag C-terminal domain (CTD), which harbors three clusters of glycine and arginine residues named GR boxes (GRI-III). In this study we investigated how PFV Gag subnuclear distribution might be regulated.

Authors : Paris Joris, Tobaly-Tapiero Joëlle, Giron Marie-Lou, Burlaud-Gaillard Julien, Buseyne Florence, Roingeard Philippe, Lesage Pascale, Zamborlini Alessia, Saïb Ali,



(9) [Application of Chimeric Antigen Receptor-Modified NK Cells in Multiple Myeloma].[TOP]

Pubmed ID :29950222
Publication Date : //
To explore the killing effect of CAR (CD138-CD28-CD3ζ)-NK cells on myeloma cells through construction of CAR(CD138-CD28-CD3)-NK cells.

Authors : Wei Hua-Ping, Yang Nan, Gu Zhen-Yang, Zhao Sha-Sha, Wang Fei-Yan, Luo Lan, Guan Li-Xun, Gao Chun-Ji,



(10) Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77 Expressed in Prokaryotic and Eukaryotic Cells.[TOP]

Pubmed ID :29912170
Publication Date : //
The mouse mammary tumor virus (MMTV) Pr77 polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77-His₆-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77-His₆-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His₆-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77-His₆-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77 should lay down the foundation towards performing RNA⁻protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.

Authors : Chameettachal Akhil, Pillai Vineeta Narayana, Ali Lizna Mohamed, Pitchai Fathima Nuzra Nagoor, Ardah Mustafa Taleb, Mustafa Farah, Marquet Roland, Rizvi Tahir Aziz,