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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) Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty.[TOP]

Pubmed ID :30852270
Publication Date : //
Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 10 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.

Authors : Berg Karen, Schäfer Vanessa Nicole, Bartnicki Natalie, Eggenschwiler Reto, Cantz Tobias, Stitz Jörn,



(2) In vitro study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia.[TOP]

Pubmed ID :30698159
Publication Date : //
Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.

Authors : Farajifard Hamid, Zavvar Mahdi, Rajaei Taraneh, Noorbakhsh Farshid, Nikougoftar-Zarif Mahin, Azadmanesh Kayhan, Kompani Farzad, Rezaei Nima,



(3) A protocol for generating induced T cells by reprogramming B cells .[TOP]

Pubmed ID :30671224
Publication Date : //
Obtaining T cells by reprogramming is one of the major goals in regenerative medicine. Here, we describe a protocol for generating functional T cells from Hoxb5-expressing pro/pre-B cells . This protocol includes the construction of Hoxb5 recombinant plasmids, retroviral packaging, isolation and viral transduction of pro/pre-B cells, cell transplantation, and phenotypic analysis of induced T cells. The procedure is reproducible and straightforward, providing an approach for generating induced T cells for translational research.

Authors : Weng Qitong, Hu Fangxiao, Zhang Mengyun, Dong Yong, Lv Cui, Wang Ying, Liu Xiaofei, Wang Jinyong,



(4) A method of producing genetically manipulated mouse mammary gland.[TOP]

Pubmed ID :30611295
Publication Date : //
To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system.

Authors : Tagaya Hiroaki, Ishikawa Kosuke, Hosokawa Yoshito, Kobayashi Shun, Ueoka Yukino, Shimada Mayuna, Ohashi Yasuko, Mikami Hirofumi, Yamamoto Mizuki, Ihara Tatsuya, Kumazawa Kentaro, Sugihara Kosuke, Goshima Naoki, Watanabe Shinya, Semba Kentaro,



(5) Standardized Method for the Study of Antibody Neutralization of HCV Pseudoparticles (HCVpp).[TOP]

Pubmed ID :30593644
Publication Date : //
Hepatitis C virus (HCV) pseudoparticles (HCVpp) are generated by cotransfection of HCV envelope (E1 and E2) genes along with a retroviral packaging/reporter construct into HEK293T cells. Enveloped particles bearing HCV E1E2 proteins on their surface are released through a retroviral budding process into the supernatant. Viral E1E2 glycoproteins facilitate a single round of receptor-mediated entry of HCVpp into hepatoma cells, which can be quantified by reporter gene expression. These HCVpp have been employed to study mechanisms of HCV entry into hepatoma cells, as well as HCV neutralization by immune sera or HCV-specific monoclonal antibodies.

Authors : Bailey Justin R, Urbanowicz Richard A, Ball Jonathan K, Law Mansun, Foung Steven K H,



(6) A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.[TOP]

Pubmed ID :30520725
Publication Date : //
Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized.

Authors : OhAinle Molly, Helms Louisa, Vermeire Jolien, Roesch Ferdinand, Humes Daryl, Basom Ryan, Delrow Jeffrey J, Overbaugh Julie, Emerman Michael,



(7) 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,



(8) Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization.[TOP]

Pubmed ID :30217825
Publication Date : //
Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

Authors : Wu Weixin, Hatterschide Joshua, Syu Yu-Ci, Cantara William A, Blower Ruth J, Hanson Heather M, Mansky Louis M, Musier-Forsyth Karin,



(9) Lentiviral-mediated BCL2 gene knockdown using comparative microRNA adaptive shRNAs.[TOP]

Pubmed ID :30213285
Publication Date : //
B-cell lymphoma 2 (BCL2) family proteins play a critical role in tuning cell death processes. Almost in half of all human cancers, a dysregulation in BCL2 family gene expression has been shown which made it an impressive target for human gene therapy as a novel approach in cancers. In this study we will optimize lentiviral-mediated RNA interference (RNAi), recombinant lentiviruses accommodating anti-BCL2 micro adaptive short hairpin RNAs (shRNAs), to downregulate BCL2 in human embryonic kidney 293T (HEK293T) cells to produce stable cell lines. We tested 4 different Dharmacon™ GIPZ™ shRNAmir lentiviral vectors targeting BCL2 in different positions and a pGIPZ non-silencing shRNAmir lentiviral vector (as a negative control). Lentivirus packaging was performed by the calcium phosphate precipitation method. HEK293T cells were transduced by each type of recombinant lentiviruses individually and selected by puromycin within 10 days. The relative mRNA level and protein expression were assayed by using real-time polymerase chain reaction (PCR) technic and western blotting, respectively. Lentivirus (LV) packaging was performed in high efficiency (transfection rate was > 90%). Recombinant viruses of 4 expression vector addition to a control vector were produced then transduced to HEK293T cells successfully. All the 4 cell groups showed a significant down regulation of BCL2 gene (~90-95%) at mRNA level compared to the control group (p<0.01) but differences between silenced groups were not significant (P > 0.05). We showed that the lentivirus-mediated RNAi technique is an efficient method to establish HEK293 cell lines with stable down-regulation of BCL2 gene.

Authors : Abdolhossein Zadeh Baharak, Yavari Kamal, Banan Mehdi, Fallah Ali, Nasehi Leila, Absalan Moloud, Tavoosidana Gholamreza,



(10) 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,