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  1. Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line
    Nordin F, Hamid ZA, Chan L, Farzaneh F, Hamid MK
    Methods Mol Biol, 2016;1448:159-73.
    PMID: 27317180 DOI: 10.1007/978-1-4939-3753-0_12
    Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.
    Matched MeSH terms: Lentivirus/genetics*
  2. Transgene expression from CpG-reduced lentiviral gene delivery vectors in vitro
    Low PT, Lai MI, Ngai SC, Abdullah S
    Gene, 2014 Jan 1;533(1):451-5.
    PMID: 24120896 DOI: 10.1016/j.gene.2013.09.075
    Current viral gene delivery vectors for gene therapy are inefficient due to short-lived transgene expression attributed to the cytosine-phosphate-guanine (CpG) motifs in the transgene. Here we assessed the effects of CpG motif reduction in lentiviral (LV) gene delivery context on the level and duration of reporter gene expression in Chinese Hamster Ovary (CHO) cells, Human Immortalized Myelogenous Leukemia (K562) cells and hematopoietic stem cells (HSCs). The cells were transduced with LV carrying Zero-CpG green fluorescent protein (ZGFP) reporter gene, LV/CMV/ZGFP. The GFP expression was compared to its non CpG-depleted GFP reporter gene LV (LV/CMV/GFP) counterpart. The LV/CMV/ZGFP exhibited prolonged transgene expression in CHO cells and HSCs up to 10 days and 14 days, in the respective cells. This effect was not seen in the transduced K562 cells, which may be due to the DNA hypomethylation status of the cancer cell line. Transgene copy number analysis verified that the GFP expression was not from pseudo-transduction and the transgene remained in the genome of the cells throughout the period of the study. The modest positive effects from the LV/CMV/ZGFP suggest that the reduction of CpG in the LV construct was not substantial to generate higher and more prolonged transgene expression.
    Matched MeSH terms: Lentivirus/genetics*
  3. Fulltext Endogenous lentivirus in Malayan colugo (Galeopterus variegatus), a close relative of primates
    Hron T, Fábryová H, Pačes J, Elleder D
    Retrovirology, 2014;11:84.
    PMID: 25280529 DOI: 10.1186/s12977-014-0084-x
    A significant fraction of mammalian genomes is composed of endogenous retroviral (ERV) sequences that are formed by germline infiltration of various retroviruses. In contrast to other retroviral genera, lentiviruses only rarely form ERV copies. We performed a computational search aimed at identification of novel endogenous lentiviruses in vertebrate genomes.
    Matched MeSH terms: Lentivirus/classification*; Lentivirus/genetics*
  4. Current techniques in reprogramming cell potency
    Romli F, Alitheen NB, Hamid M, Ismail R, Abd Rahman NM
    J Cell Biochem, 2013 Jun;114(6):1230-7.
    PMID: 23239017 DOI: 10.1002/jcb.24477
    The first successful attempt to reprogram somatic cell into embryonic-like stem cell was achieved on 2006. Since then, it had sparked a race against time to bring this wonderful invention from bench to bedside but it is not easily achieved due to severe problems in term of epigenetic and genomic. With each problem arise, new technique and protocol will be constructed to try to overcome it. This review addresses the various techniques made available to create iPSC with problems hogging down the technique.
    Matched MeSH terms: Lentivirus/genetics
  5. Fulltext A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
    Kalidasan V, Ng WH, Ishola OA, Ravichantar N, Tan JJ, Das KT
    Sci Rep, 2021 Sep 28;11(1):19265.
    PMID: 34584147 DOI: 10.1038/s41598-021-98657-7
    Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.
    Matched MeSH terms: Lentivirus/genetics*
  6. Current viral-mediated gene transfer research for treatment of Alzheimer's disease
    Sasmita AO
    Biotechnol Genet Eng Rev, 2019 Apr;35(1):26-45.
    PMID: 30317930 DOI: 10.1080/02648725.2018.1523521
    Alzheimer's disease (AD) is the most common form of dementia and has affected millions of individuals worldwide. The hallmarks of AD include the amyloid beta plaque deposits, tau neurofibrillary tangles, altered neuronal signaling, alongside decline in memory and cognitive functions. Conventional drug therapies do exist, such as donepezil or aducanumab, but these drugs mostly focus on halting AD progression instead of causing a reversal within the disease. In an effort to ameliorate and ultimately cure AD, researchers have delved into viral-mediated gene therapy to fix this disease from its root molecular causes. To date, adeno-associated virus and lentiviral vectors have remained the most vastly studied among other viral vectors to combat AD. These vectors could be employed alongside various genetic materials based on the types of processes we want to alter to yield a positive effect, such as disruption of amyloidogenic pathway, neuroprotection and lipid metabolism pathways. Recent studies and trials were reviewed in this article, highlighting their clinical significance, differences and limitations between each method. By learning from the different combinations and possibilities of viral-mediated gene transfer, researchers would then get a step closer in ameliorating symptoms and possibly in curing AD.
    Matched MeSH terms: Lentivirus
  7. Fulltext Production of Lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
    Siew, Ching Ngai, Ramasamy, Rajesh, Syahril Abdullah
    Pertanika Journal of Science & Technology, 2012;20(2):269-281.
    MyJurnal
    Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike nonviralmethods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome,which can lead to long-term transgene expression. Lentiviruses have advantages over other types ofviruses due to their capacity to transduce non-dividing cells. An optimized generation of lentivirusescarrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) orCMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by cotransfectinglentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines.Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine(PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvestedat days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFPharvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally,the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transientand stable GFP expression. It was found that the GFP expression declined overtime. However, thetransduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentivirusescarrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells atdifferent efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to proceed to the subsequent analysis of the effect of regulatory elements in future study.
    Matched MeSH terms: Lentivirus
  8. Fulltext Seroprevalence of small ruminant caprine arthritis encephalitis lentivirus among goats from selected small ruminant farms in Selangor, Malaysia
    Jesse FFA, Bitrus AA, Abba Y, Raju VN, Hambali IU, Peter ID, et al.
    Vet World, 2018 Feb;11(2):172-176.
    PMID: 29657399 DOI: 10.14202/vetworld.2018.172-176
    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.

    Materials and Methods: Blood samples were collected from a total of 91 goats selected at random. Blood serum was harvested and used for competitive enzyme-linked immunosorbent assay test to detect antibodies against CAE virus.

    Results: The result obtained showed that 8/91 (8.8%) of the goats were seropositive for CAEV. In addition, biosecurity management, source of origin and sex of the animal were observed to be important risk factors associated with the occurrence of CAE in goats.

    Conclusion: The findings of this study affirmed that the seroprevalence of CAEV infection among goat population in small ruminant farms in Selangor, Malaysia, is low. However, there is need to institute strict control measures such as testing and culling positive animals or separation of infected animals from those that tested negative to the disease for effective eradication of the disease.

    Matched MeSH terms: Lentivirus Infections
  9. A novel rat model of Alzheimer's disease based on lentiviral-mediated expression of mutant APP
    Parsi S, Pandamooz S, Heidari S, Naji M, Morfini G, Ahmadiani A, et al.
    Neuroscience, 2015 Jan 22;284:99-106.
    PMID: 25270904 DOI: 10.1016/j.neuroscience.2014.09.045
    Alzheimer's disease (AD) is characterized by progressive and irreversible cognitive and memory impairment. The discovery of familial forms of AD (fAD) in association with specific gene mutations facilitated the generation of numerous rodent models. These models in turn proved valuable for the study of molecular mechanisms underlying AD pathogenesis, and facilitated translational research and preclinical drug development. This study aimed to introduce a new rat model of AD simulating some aspects of the sporadic cases of disease.
    Matched MeSH terms: Lentivirus/genetics
  10. Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression
    Ngai SC, Rosli R, Nordin N, Veerakumarasivam A, Abdullah S
    Gene, 2012 May 1;498(2):231-6.
    PMID: 22366305 DOI: 10.1016/j.gene.2012.01.071
    Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.
    Matched MeSH terms: Lentivirus/genetics*
  11. Fulltext Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer
    Škalamera D, Dahmer-Heath M, Stevenson AJ, Pinto C, Shah ET, Daignault SM, et al.
    Oncotarget, 2016 Sep 20;7(38):61000-61020.
    PMID: 27876705 DOI: 10.18632/oncotarget.11314
    Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy.
    Matched MeSH terms: Lentivirus/metabolism
  12. Fulltext DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells
    Ngai SC, Rosli R, Al Abbar A, Abdullah S
    Biomed Res Int, 2015;2015:346134.
    PMID: 25961011 DOI: 10.1155/2015/346134
    Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.
    Matched MeSH terms: Lentivirus/genetics
  13. Fulltext Enhanced Contextual Fear Memory and Elevated Astroglial Glutamate Synthase Activity in Hippocampal CA1 BChE shRNA Knockdown Mice
    Chen S, Lin Z, Tan KL, Chen R, Su W, Zhao H, et al.
    Front Psychiatry, 2020;11:564843.
    PMID: 33061920 DOI: 10.3389/fpsyt.2020.564843
    Butyrylcholinesterase (BChE) efficiently hydrolyzes acetylcholine (ACh) at high concentrations when acetylcholinesterase (AChE) is substrate-inhibited. Recent studies have shown that BChE also has a function that is independent of ACh, but it has not been fully explored. Low BChE expression is accompanied with higher stress-induced aggression and ghrelin levels in stress models, and BChE knockout mice exhibit cognitive and memory impairments. However, the role of BChE in posttraumatic stress disorder (PTSD) remains unclear. In the present study, we investigated the role of BChE in contextual fear memory and its regulatory effect on the expression of factors related to the glutamate (Glu)-glutamine (Gln) cycle via knockdown studies. We used AAVs and lentiviruses to knockdown BChE expression in the mouse hippocampal CA1 region and C8D1A astrocytes. Our behavioral data from those mice injected with AAV-shBChE in the hippocampal CA1 region showed strengthened fear memory and increased dendritic spine density. Elevated Glu levels and glutamine synthetase (GS) enzyme activity were detected in contextual fear conditioned-BChE knockdown animals and astrocytes. We observed that an AAV-shBChE induced lowering of BChE expression in the hippocampus CA1 region enhanced contextual fear memory expression and promoted the astrocytic Glu-Gln cycle but did not elevate ACh-hydrolyzing activity. This study provides new insight into the regulatory role of BChE in cognition and suggests potential target for stress-related psychiatric disorder such as PTSD where patients experience fear after exposure to severe life-threatening traumatic events.
    Matched MeSH terms: Lentivirus
  14. Overcoming the challenge of transduction of human T-cells with chimeric antigen receptor (CAR) specific for ERBB2 antigen
    Munisvaradass R, Ding SSL, Ee AHK, Syahril Abdullah, Mok PL, Kumar S, et al.
    Sains Malaysiana, 2017;46:1831-1838.
    Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the
    overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor
    (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is
    specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are
    able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering
    it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human
    T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction
    protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene.
    CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted
    into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These
    lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabeadconjugated
    CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected
    and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models
    to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells.
    Matched MeSH terms: Lentivirus
  15. Fulltext AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation
    Cullen JK, Abdul Murad N, Yeo A, McKenzie M, Ward M, Chong KL, et al.
    PLoS One, 2016;11(2):e0148213.
    PMID: 26866375 DOI: 10.1371/journal.pone.0148213
    Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.
    Matched MeSH terms: Lentivirus/genetics
  16. Fulltext Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells
    Munisvaradass R, Kumar S, Govindasamy C, Alnumair KS, Mok PL
    Int J Mol Sci, 2017 Sep 08;18(9).
    PMID: 28885562 DOI: 10.3390/ijms18091797
    Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non-transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.
    Matched MeSH terms: Lentivirus/genetics
  17. Fulltext Optogenetic control of iPS cell-derived neurons in 2D and 3D culture systems using channelrhodopsin-2 expression driven by the synapsin-1 and calcium-calmodulin kinase II promoters
    Lee SY, George JH, Nagel DA, Ye H, Kueberuwa G, Seymour LW
    J Tissue Eng Regen Med, 2019 Mar;13(3):369-384.
    PMID: 30550638 DOI: 10.1002/term.2786
    Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.
    Matched MeSH terms: Lentivirus
  18. Fulltext Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation
    Greenwood MP, Greenwood M, Mecawi AS, Antunes-Rodrigues J, Paton JF, Murphy D
    Mol Brain, 2016 Jan 07;9:1.
    PMID: 26739966 DOI: 10.1186/s13041-015-0182-2
    BACKGROUND: Rasd1 is a member of the Ras family of monomeric G proteins that was first identified as a dexamethasone inducible gene in the pituitary corticotroph cell line AtT20. Using microarrays we previously identified increased Rasd1 mRNA expression in the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in response to increased plasma osmolality provoked by fluid deprivation and salt loading. RASD1 has been shown to inhibit adenylyl cyclase activity in vitro resulting in the inhibition of the cAMP-PKA-CREB signaling pathway. Therefore, we tested the hypothesis that RASD1 may inhibit cAMP stimulated gene expression in the brain.

    RESULTS: We show that Rasd1 is expressed in vasopressin neurons of the PVN and SON, within which mRNA levels are induced by hyperosmotic cues. Dexamethasone treatment of AtT20 cells decreased forskolin stimulation of c-Fos, Nr4a1 and phosphorylated CREB expression, effects that were mimicked by overexpression of Rasd1, and inhibited by knockdown of Rasd1. These effects were dependent upon isoprenylation, as both farnesyltransferase inhibitor FTI-277 and CAAX box deletion prevented Rasd1 inhibition of cAMP-induced gene expression. Injection of lentiviral vector into rat SON expressing Rasd1 diminished, whereas CAAX mutant increased, cAMP inducible genes in response to osmotic stress.

    CONCLUSIONS: We have identified two mechanisms of Rasd1 induction in the hypothalamus, one by elevated glucocorticoids in response to stress, and one in response to increased plasma osmolality resulting from osmotic stress. We propose that the abundance of RASD1 in vasopressin expressing neurons, based on its inhibitory actions on CREB phosphorylation, is an important mechanism for controlling the transcriptional responses to stressors in both the PVN and SON. These effects likely occur through modulation of cAMP-PKA-CREB signaling pathway in the brain.

    Matched MeSH terms: Lentivirus/metabolism
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