Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells / 中南大学学报(医学版)
Journal of Central South University(Medical Sciences)
;
(12): 8-15, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-814132
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Osteochondrodysplasias
/
Molecular Sequence Data
/
Base Sequence
/
Transfection
/
Chlorocebus aethiops
/
Mutagenesis, Site-Directed
/
Insulin-Like Growth Factor Binding Proteins
/
COS Cells
/
CCN Intercellular Signaling Proteins
/
Genetics
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Journal of Central South University(Medical Sciences)
Year:
2008
Type:
Article
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