コクラ　ケンジ   Kokura Kenji   古倉　健嗣    所属   東邦大学  理学部 生物分子科学科    職種   准教授
言語種別	英語
発表タイトル	Establishment of reporter cell lines to monitor Epithelial-Mesenchymal Transition
会議名	第77回日本細胞生物学会
主催者	日本細胞生物学会
学会区分	国内学会
発表形式	ポスター掲示
講演区分	一般
発表者・共同発表者	Rin Fukumoto, Kaya Osanai, Konoka Nakamura, Toshihiko Kishimoto, Kenji Kokura
発表年月日	2025/07/16
開催地 （都市, 国名）	名古屋
概要	Epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells acquire mesenchymal traits in response to stimuli such as inflammation or cytokines. E- cadherin and keratin are typical epithelial markers, while N-cadherin and vimentin indicate mesenchymal states. EMT is crucial for cancer invasion and metastasis, but completion of metastasis also requires mesenchymal-epithelial transition(MET), the reverse process of EMT. Therefore, establishing an experimental system that enables time-lapse observation of both EMT and MET processes is essential for studying cancer metastasis.To study EMT and MET dynamics, we aimed to visualize E-cadherin and N- cadherin expression using fluorescent proteins in live cells. We generated reporter cell lines by inserting florescent tags at the C-termini of the E-cadherin and N-cadherin genes via CRISPR-Cas9. To reduce off-target effects, we applied the double nickase method and carefully designed guide RNAs, enabling efficient knock-in.Using A549 human lung cancer cells, we established fluorescent reporter lines with green fluorescent proteins fused to the endogenous E-cadherin and N-cadherin proteins. Expression was validated by western blot and fluorescence microscopy.Additionally, for N-cadherin, we are applying the Cre- LoxP system to remove the PGK-driven drug resistance gene.