[Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells].
OBJECTIVE
To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells.


METHODS
Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot.


RESULTS
By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells.


CONCLUSIONS
WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.
Wang
Min
M
Institute of Metablism and Endocrinology, Second Xiangya Hospital, Central South University, Changsha 410011,China.
Peng
Yi-qun
YQ
Zhou
Hou-de
HD
Zhai
Mu-xu
MX
He
Yu-ling
YL
Xie
Hui
H
D000818:Animals; D001483:Base Sequence; D055511:CCN Intercellular Signaling Proteins; D019556:COS Cells / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010009:Osteochondrodysplasias / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D055511:CCN Intercellular Signaling Proteins; C400081:CCN6 protein, human; D018969:Insulin-Like Growth Factor Binding Proteins