Construction Of P120 Knock Out Cell Line Using CRISPR/Cas9 System

Cell junctions(or intercellular bridge)are structures that exist within the tissue of some multicellular organisms.Cell junctions consist of multiprotein complexes that provide contact between neighboring cells or between a cell and the extracellular matrix.They also build up the paracellular barrier of epithelia and control the paracellular transport.Cell junctions are especially abundant in epithelial tissues.Cell junctions are especially important in enabling communication between neighboring cells via specialized proteins called communicating junctions.Cell junctions are also important in reducing stress placed upon cells.Epithelial cells are in close contact with each other by tight junctions,adherens junctions and gap junctions that connect these cells.In the epithelial cell to mesenchymal cell transformation process,the cell and cell connection will gradually dissociate.With the close connection and the dissociation of the attachment structure of the dissociation,epithelial cell morphology and phenotype have changed,the cells lost close growth status,showing sparse,loose growth,cells showed spindle shape.There are more studies on tight junctions,and relatively few for adherent connections.In this paper,the protein of p120 in NMUMG of mouse mammary epithelial cells was knocked out specifically by using CRISPR/Cas9 technique,and p120 stable knockout cell line was constructed.It was found that NMUMG cells lost close growth after knockout of p120,and showed a loose growth state,and the cell morphology changed.And then by replenishing the wild type p120,NMUMG was found to be able to return to normal cell growth.This proves that p120 is essential for NMUMG to maintain normal cell morphology.Through immunofluorescence experiments,it can be seen that E-cadherin is dissociated from the cell membrane after p120 knockout,and E-cadherin is a symbolic protein attached to adhesion,which means that the disappearance of adhesion between NMUMG cells Indicating that p120 can stabilize E-cadherin,promote the formation of adhesion between epithelial cells.And for p120 is how to stabilize E-cadherin to control the adhesion of the connection,pending further study.