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Identification and Characterisation of Prohibitin-2 as a Receptor for Dengue Virus in Insect Cell

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Added on  2019-10-18

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This article discusses the identification and characterisation of Prohibitin-2 as a receptor for Dengue virus in insect cell using molecular and cellular techniques. It explains how CRISPR/Cas9 was used to knock out the Prohibitin-2 gene and study the dengue virus-host interaction. The article also outlines the study design and methodology, including cell culture, DNA extraction, PCR, gel electrophoresis, and DNA sequencing.

Identification and Characterisation of Prohibitin-2 as a Receptor for Dengue Virus in Insect Cell

   Added on 2019-10-18

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Abstract Dengue is a mosquito-borne viral disease. Dengue incidence has increased more than 30-fold in recent decades due to the geographical expansion of the Aedes vector mosquitoes and dengue viruses. Thus, vector control is a fundamental approach to reduce disease transmission and spread. Identification of dengue receptors is critical in understanding Dengue life cycle then control the infection transmission. A putative dengue virus receptor, Prohibitin-2, has been successfully knocked out in C6/36 cell line derived from mosquitos. An insect vector has been used to express a sgRNA complementary to Prohibitin-2 gene of Aedes albopictus mosquito using CRISPR/Cas9 vector. Then the DNA plasmid has been transfected into a C6/36 cell line. Therefore, C6/36 cells with a knockout in Prohibitin-2 is selected, expanded, and Grown in cell culture to study the dengue virus-host interaction among singlecells. This project purpose is to created and characterised insect cell lines for genetic knockout of the putative dengue receptor, prohibitin-2 using molecular and cellular techniques. We characterised cell population of two distinct cell lines, Odd3 and 4G5 cell lines, derived from parent C6/36 cell. The characterisation concluded achieving mono-allelic knockdown in Odd3 cell line. Thus, Odd3 is re-edited using CRISPR/Cas9 to achieve mutation on the other allele. The characterisation of cells derived from Odd3 cell line after re-editing, Odd3-4E4 and Odd3-1C3 suggested retaining the allelic mutation in Odd3 cells. On the other hand, 4G5 cell line characterisation revealed inheritance of in-frame deletions of 24 amino acids that do not cause loss of prohibition-2 function.7. Aims and objectives Prohibitin is identified and characterised as a receptor, and interacting protein mediated Dengue virus serotype-2 entry (Kuadkitkan et al., 2010). The study utilised insect cell line derived from Aedes mosquitoes. Using VOPBA analysis, Prohibitin, conserved and expressed in the vast majority of eukaryotic cells, was identified when segregatedwith susceptibility to infection. Co-immunoprecipitation demonstrated the interaction between Dengue virus and Prohibitin. Moreover, the role of Prohibitin in insect cell entry was confirmed by small interfering RNA (siRNA), and antibody mediated infection inhibition as well as colocalization of the virus and protein on the cell surface. The characterisation of the interaction was only with dengue virus serotype-2. Thus, there is a possibility of interacting with other dengue serotypes.Therefore, we are aiming to identify and characterise prohibitin-2 as a receptor for the dengue virus in insect cell via several stringent molecular and cellular techniques. Using CRISPR/Cas9, Dr Shiu-Wan has had knocked out the prohibitin-2 gene to study the dengue virus-host interaction. Also, we are aiming to prove that the Prohibition-2 gene can be silenced using CRISPR/Cas9 genome manipulation tool which affects the functional role of Prohibition-2 gene. Prohibition-2 wild type must convert to bi-allelic mutation to achieve successful gene function silencing. A successful Prohibition-2 gene silencing is critical in control the spreading and transmission of DENV by controlling thevector, Aedes mosquitoes.This project is a part of the main project started by Dr Shiu-Wan. An insect vector has been used to express a sgRNA complementary to Prohibitin-2 gene of Aedes albopictus mosquito using CRISPR/Cas9 vector. Then the DNA plasmid has been transfected into a C6/36 cell line. Therefore, C6/36 cells with a knockout in Prohibitin-2 is selected by limited dilution, expanded, and Grown in cell culture to study the dengue virus-host interaction among single cells. Hence, the objectives of this project are to create and maintain cell lines derived from C6/36 that previously knocked out, and characterise these cells populations for genetic knockout of the putative dengue receptor, prohibitin-2. After cell culture, DNA is extracted, amplified by PCR, and run on gel electrophoresis to identify INDELS that will be confirmed by DNA sequencing.
Identification and Characterisation of Prohibitin-2 as a Receptor for Dengue Virus in Insect Cell_1
8. STUDY DESIGN AND METHODOLOGY:8.1. Study Design: This project had been started by Dr Shiu-Wan Chan before we started, and we participated in the identification and characterisation using cellular and molecular techniques. To provide an overview of project workflow (Figure 4), the study design is described in three parts: the part done by DR Shiu-Wan Chan, that part done by Master students, andfuture suggestions. The first part includes the following techniques and methods:First, the cloning of sgRNA into plasmid vectors (E. coli). The vectors encode the Puromycin resistance gene (as a selectable marker to indicate the success of a transfection), cas9. Secondly, transfection of the plasmid vector by using a lipid. The cells continue to grow and passage maintaining selection pressure by keeping puromycin in the growth medium (with and without puromycin). After 1-2 weeks, a large number of the cells were killed by the puromycin, indicating that they did not take up or had lost the plasmid with the puromycin resistance gene. The cellsthat remained growing in the puromycin-containing medium had retained the expression plasmid, which may have stably integrated into the genome of the targeted cells.The third stage is clonal selection: Select clonal populations of cells by transferring a well-isolated single clump of cells (the clonal ancestor and cells derived from it) into a well in a 24 well plate; repeat to select 5-10 clonal populations. Fourth, identify a single clone through limited dilution and expansion. Limited dilution has been used with cells both with and without puromycin selection to isolate each cell that carries INDELS by placing them at very low cell densities (<1 cell per well in 96 well plates), and expanding colonies from those single cells in separate wells (24 wells then six wells plate). Mono-allelic knockout cells have been created (2nd generation). A second CRISPR knockout has been performed on these mono-allelic knockout cells to create bi-allelic knockout. This project is to screen the cloned cells, which are 3rdgeneration cells, using cellular and molecular techniques, including cell culture, DNA lysate and extraction, PCR, and gel electrophoresis as well as DNA sequencing for the bi-allelic knockout. The final part of the study depends on the success of the second part, which uses techniques such aswestern blot and immunofluorescence assay to confirm the gene silencing as well as to prove the hypothesis.
Identification and Characterisation of Prohibitin-2 as a Receptor for Dengue Virus in Insect Cell_2

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