Use of the SST-REX method for the identification of genes expressed at the condensation stage of chondrogenic cell line ATDC5.
In endochondral ossification, chondrogenesis precedes bone development. Cartilage differentiation is initiated by the formation of chondrogenic cell condensates. Thus, it is essential to investigate genes expressed at the condensation stage for a better understanding of chondrogenesis and ossification. To this end, we constructed a cDNA library from the mRNA fraction derived from a chondrogenic cell line (ATDC5) at the cell condensation stage by using the signal sequence trap by retrovirus-mediated expression (SST-REX) method. We obtained 486 factor (IL-3)-independent clones by screening 5.7 x 10(3) clones. DNA sequencing analysis of the clones identified genes encoding 157 known proteins and 4 novel proteins. These 4 genes encoding novel proteins were expressed not only in chondrogenic ATDC5 cells but also in osteogenic MC3T3-E1 and myogenic C2C12 cells. The mRNA expression level of 1 of the 4 clones increased at the calcification stage of the differentiation of MC3T3-E1 cells. The results demonstrate that the SST-REX method is a useful experimental system to identify genes involved in the complicated mechanisms of bone formation.
Noguchi
Atsushi
A
Research Institute for Biological Sciences, Tokyo University of Science (Rikadai), 2669 Yamazaki, Noda, 278-0022, Japan.
Watanabe
Naoko
N
Fujimaki
Ryoji
R
Kitamura
Toshio
T
Hayashizaki
Yoshihide
Y
Miyaki
Shigeru
S
Tezuka
Kenichi
K
Hozumi
Nobumichi
N
D000818:Animals; D015152:Blotting, Northern; D001842:Bone and Bones / Q000378:metabolism; D002356:Cartilage / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D019902:Chondrocytes / Q000378:metabolism; D020219:Chondrogenesis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D007377:Interleukin-3 / Q000378:metabolism; D051379:Mice; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics
D018076:DNA, Complementary; D007377:Interleukin-3; D012333:RNA, Messenger; D012313:RNA