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Prohibitin-2 | Receptor of Dengue Virus

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Added on  2019-10-08

Prohibitin-2 | Receptor of Dengue Virus

   Added on 2019-10-08

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Aims and objectives We are aiming to identify and characteriseprohibitin-2 as a receptor of Dengue virus in insect via several stringent molecular and cellular techniques. CRISPR/Cas9 has been used to Knockout the prohibitin-2 gene to study the virus-Host interaction and prove that PHB-2 is a receptor for Dengue virus ininsect cells. Also, we are aiming to prove that PHB-2 gene can be silenced to stop their functional role by using CRISPR/Cas9 tools to manipulate the PHB-2 gene, resulting in mutation in the genomic sequence. To achieve successful gene function silencing, bi- allelic mutation must occur. Successful PHB-2 silencing in insect cells will stop Dengue virus entering insect cells. Consequently, Dengue virus will be manageable as a result of controlling of the vector spreading. Research Design Overview: This project has been started by Dr Shiu-Wan Chan before we begin, which we participate in the identification and characterization by using few techniques and we did not finish the whole project due to dissertation project time. To provide an overview of project workflow (Figure), the study design is described in three parts: part done by DR Shiu-Wan, by Master students as well as future suggestion. The first part includes the following techniques and methods:Firstly, Cloning of SgRNA into plasmid Vector (E. coli). The vectors encode Puromycin resistance gene (as Aselectable marker to indicate the success of atransfection), cas9. Secondly, Transfection of plasmid vector by using lipid. Continue to grow and passage the cells maintaining selection pressure by keeping puromycin in the growth medium (with and without puromycin). After 1-2 weeks, a large number of the cells will be killed by the puromycin, indicating that they did not take up or have lost the plasmid with the puromycin resistance gene. The cells that remain growing in the puromycin-containing medium have retained the expression plasmid, which may have stably integrated into the genome of the targeted cells. Thirdly, 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 of a 24 well plate; repeat to select 5-10 clonal populations. Fourthly, Identify single cloned by limited dilution and expansion. Limited dilution has been used with cells with and without puromycin selection to isolate each cell that carries INDELS by placing them at very low cell densities (< 1 well per well in 96 well plates), and expand colonies from those single cells in separate wells (24 wells then six wells plate).
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