Virology2Aims and objectivesSeveral stringent cellular and molecular techniques have been used to identify and portray the prohibitin-2 as a receptor of infection of Dengue in a bug. In order to study the virus namely the Host interaction and thereby proving that PHB-2 is a receptor of Dengue virus in the cells of the insects CRISPR/Cas9 is being used to knockout the prohibitin-2 gene. Likewise in order to manipulate the PHB-2 gene, which would result in the mutation of genomic sequence, there has been an aim to prove that PHB-2 gene could also be silenced just to stop the functional roles with the help of CRISPR/Cas9 tools. Bi-allelic mutation must happen to accomplish the effective gene function silencing. For stopping the virus namely Dengue to enter the insect cells will definitely silence the PHB-2. Thus, the infection of dengue would be reasonable because of controlling of the vector being spread. Research Design Overview:Dr. Shin-Wan Chan was the person to start this project, and this project is for the characterization as well as identification with the help of few techniques. For providing an overview of the project workflow, the study has been designed in three parts namely- parts done by Dr. Shiu-Wan and few of the stuents pursuingMasters. The part one of the study includes- Firstly, plasmid vector namely (E-coli) cloned into SgRNA. Puromycin resistance gene is the vector that is encoded Cas9 which act as a selectable marker for the indication of transfection. On the second hand, Using lipid by the transfection of plasmid vector. These help in the continuous growth and helps the cells maintain the selection pressure thereby making the puromycin in the growth medium. Puromycin helps in the killing of large quantity of cells thereby indicating that the plasmid is lost with puromycin resistance gene, this takes places after one to two weeks of the first step. Retention of the plasmid is through the cells that remain growing in the puromycin containing medium and this may have stably integrated into the genome of the targeted cells.
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