Vol. 44 No. 2 SCIENCE IN CHINA (Series C) April 2001 Establishment of murine Smad5 double knockout ES cells and the studies on their properties YANG Xiao (杨 晓) 1 , SUN Yanxun (孙彦洵) 1 , ZHOU Jiang (周 江) 1 ， HUANG Peitang (黄培堂) 1 , HUANG Cuifen (黄翠芬) 1 , XU Xiaoling (徐晓玲) 2 , LI Cuiling (李翠玲) 2 , Gotay Jessica 2 , CHEN Lin (陈 林) 2 & DENG Chuxia (邓初夏) 2 1. Institute of Biotechnology, Beijing 100071, China; 2. National Institute of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda 20892, USA Correspondence should be addressed to Yang Xiao (email: yangx@nic.bmi.ac.cn) Received June 13, 2000 Abstract Smad5 is an intracellular transducer of TGF-β signals. Targeted disruption of murine Smad5 gene resulted in embryonic lethal. To study the function of Smad5 in organgenesis, we generated Smad5 double knockout ES cells by homologous recombination. We deleted the neo gene of the Smad5 targeted ES cells using Cre-LoxP system. Smad5 double knockout ES cells were obtained by transfecting the targeted ES cells using the same targeting construct. The results of chimeric study showed that Smad5 might play an important role during the development of heart and neural tube. Smad5 double knockout ES cells formed teratoma when injected subcutaneously into nude mice. They differentiated into several types of cells, including neural cells, muscle cells, chondrocytes, endothelial cells and glandaceous cells. Smad5 double knockout ES cells are useful for studying the function of Smad5 mediated TGF- β during the organgenesis and the in vitro dif- ferentiation of ES cells. Keywords: Smad5, double knockout, ES cells, Cre-LoxP. Transforming growth factor-β (TGF-β) is a large superfamily of cytokines, including the bone morphogenetic proteins (BMPs), inhibins, activins and TGF-βs. They play an important role in the vertebrate development by regulating the cell proliferation, differentiation, adhesion, migra- tion and apoptosis. TGF-β signals through a transmembrane serine/threonine kinase receptor complex. Recently, a new gene family ⎯ Smads family has been found to be an important component of the TGF-β signal transduction pathway [1,2] . SMADs are divided into 3 classes: The first is re- ceptor activated SMADs, including SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8. They have a highly conserved phosphorylation site SS(V/M)S at the end of the C terminal domain. They can be phosphorylated directly by the type I receptors. The phosphorylated SMADs form complex with the common SMAD and translocate into nucleus to regulate the target genes to response to the TGF-β signals. The second is common SMAD. So far, the only one of this class is SMAD4 which shares relatively low homology with other members of the SMADs family. There is no