Novel method for establishing claudin knockout clones using TALENs

Abstract

In the multicellular organisms, epithelia function as a barrier between external and internal environment. Tight junctions (TJs) are one of the cell‐cell junctions between epithelial cells and regulate the permeability of the paracellular pathway. Claudins, a large family of transmembrane proteins, are major component of TJ strands. Most epithelia express multiple different claudins, and the expression pattern of claudins are thought to provide diverse permeability of TJs in each epithelium. However, it is difficult to measure the permeability of each claudin with the current technique, and the permeability property of each claudin is still poorly understood. To clarify the permeability property of each claudin, we attempted to knockout claudin genes using transcription activator‐like effector nucleases (TALENs) in MDCK cells. In the previous study, we succeeded in establishing claudin‐2 knockout clones and claudin‐4 knockout clones in MDCK cells. However, the efficiency of the knockout is low and a lot of effort was required to establish knockout clones. To improve the efficiency of establishing claudin knockout clones, we made a construct in which forward and reverse TALENs were connected with internal ribosome entry site (IRES). Using the constructs, we succeeded in establishing claudin‐2, −3, −4 and −7 knockout clones. In conclusion, we developed the method for establishing claudin knockout clones. The analysis of these claudin knockout clones are thought to provide an important insight in the permeability property of each claudin and TJ physiology.Support or Funding InformationJSPS Overseas Research FellowshipsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.