Lentivirus

Background and Aim: Caprine arthritis encephalitis (CAE) is an important viral disease of small ruminants particularly in dairy goats with severe social and economic implication. Hence, this study was designed to determine the seroprevalence of CAE virus (CAEV) among goat population in selected small ruminant farms in Selangor and the risk factors associated with the occurrence of the disease.

Lentiviral vectors have emerged over the last decade as powerful, reliable and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells, including neurons. This is why LVs are becoming the most useful and promising tools in the field of neuroscience, not only for research, but also for future gene and cell therapy approaches. Lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. These latest designs are reviewed in this unit. This unit also describes protocols for production and titration of LVs that can be implemented in a research laboratory setting, with an emphasis on standardization to improve transposability of results between laboratories.

The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaled-up from shake flaks to a 3-L bioreactor from which 1010 IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant. Biotechnol. Bioeng. 2007; 98: 789–799. © 2007 Wiley Periodicals, Inc.

Hematopoietic stem cell (HSC)-based gene therapy holds promise for the cure of many diseases. The field is now moving toward the use of lentiviral vectors (LVs) as evidenced by 4 successful clinical trials. These trials used vesicular-stomatitis-virus-G protein (VSV-G)-LVs at high doses combined with strong cytokine-cocktail stimulation to obtain therapeutically relevant transduction levels; however, they might compromise the HSC character. Summarizing all these disadvantages, alternatives to VSV-G-LVs are urgently needed. We generated here high-titer LVs pseudotyped with a baboon retroviral envelope glycoprotein (BaEV-LVs), resistant to human complement. Under mild cytokine prestimulation to preserve the HSC characteristics, a single BaEV-LV application at a low dose, resulted in up to 90% of hCD34(+) cell transduction. Even more striking was that these new BaEV-LVs allowed, at low doses, efficient transduction of up to 30% of quiescent hCD34(+) cells, whereas high-dose VSV-G-LVs w...

Objective: Myeloproliferative Neoplasms (MPN) is frequently seen as a fatal disease. Although the JAK2V617F mutation helps to understand MPN, there are still questions to be answered in the pathological development of the disease. JAK2V617F mutation is diagnosed in the most of the thrombotic cases, and its relation to endothelial cells is controversial. The effects of epigenetic factors on MPN are unknown. In this study, the effects of JAK2V617F mutation on endothelial cells (EC) and the SOCS methylations of this mutation which are epigenetically important are examined. Method: Lentiviral vectors that include JAK2V617F mutation and normal JAK2 genes and carry GFP were infected to HUVEC (Human umbilical vein endothelial cell) cell line. GFP was detected with flow cytometry and its analysis was conducted. The genomic DNA of cell population containing a certain percent of GFP was isolated. PCR was applied for methylation analysis by using a DNA which is exposed to bisulphite modification. Findings: Lipofectamine was determined to be the most productive among the different transfection agents. No morphological difference was observed upon the comparison of lentiviral vectors within JAKV617F, JAK2 and GFP vectors and uninfected ECs. No obvious difference is observed between the vitality rates of infected and uninfected ECs. It was found that among the infected and uninfected ECs, all, except from SOCS-1, without any observed difference, demonstrated an unmethyle profile. Result: No difference was detected in the morphologies and vitality rates of ECs which were either infected with lentiviral vectors or uninfected. SOCS4 methylation was tested in EC for the first time in literature. No similarity was observed between the methylation profiles of SOCS 1, 2, 3 genes and blood cells of MPN patients. In this study, it could not be concluded that the morphological effects of JAK2V617 mutation on ECs and differences in methylation profiles might be connected to thrombotic events. More detailed future studies on this field will help us in explaining this relation. Key Words: Myeloroliferative Neoplasm (MPN), Janus Kinase 2(JAK2), Endothelial, Epigenetics, Lentivirus.

JNK and ERK MAP kinases regulate cellular responses to genotoxic stress in a cell type and cell context-dependent manner. However, the factors that determine and execute JNK- and ERK-controlled stress responses are only partly known. In this study, we investigate the roles of the AP-1 components ATF3 and Fra1 in JNK- and ERK-dependent cell cycle arrest and apoptosis. We show that the anti-cancer drug cisplatin or UV light activates both JNK and ERK in human glioblastoma cells lacking functional p53. Inhibition experiments of JNK or ERK activities revealed that the ERK pathway strongly promotes cisplatin- and UV-induced apoptosis in these glioblastoma cells. Furthermore, JNK but not ERK is required for ATF3 induction, and both ERK and JNK are necessary for post-transcriptional induction of Fra1 in response to cisplatin or UV. Knock-down of ATF3 and Fra1 results in increased and decreased cisplatin-induced apoptosis, respectively, indicating that ATF3 is an anti-apoptotic JNK effector and Fra1 is a pro-apoptotic ERK/JNK effector. Knock-down experiments also revealed that ATF3 and Fra1, respectively, enhance and reduce S-phase arrest through differential modulation of the Chk1–Cdk2 pathway. Thus, we identify novel reciprocal functions of ATF3 and Fra1 in JNK- and ERK-dependent DNA damage responses.