[Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells].

Abstract

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.

CONCLUSION:

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.

• Authors: Min Wang, Yi-qun Peng, Hou-de Zhou, Mu-xu Zhai, Yu-ling He, Hui Xie
• Journal: Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences (Zhong Nan Da Xue Xue Bao Yi Xue Ban) Vol. 33 Issue 1 Pg. 8-15 (Jan 2008) ISSN: 1672-7347 [Print] China
• PMID: 18245897 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• CCN Intercellular Signaling Proteins
• CCN6 protein, human
• Insulin-Like Growth Factor Binding Proteins

Topics

• Animals
• Base Sequence
• CCN Intercellular Signaling Proteins
• COS Cells (metabolism)
• Chlorocebus aethiops
• Humans
• Insulin-Like Growth Factor Binding Proteins (biosynthesis, genetics)
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• Mutation
• Osteochondrodysplasias (genetics, metabolism)
• Transfection