Construction of a recombinant eukaryotic expression plasmid containing human calcitonin gene and its expression in NIH3T3 cells.
OBJECTIVE
To construct a recombinant eukaryotic expression plasmid containing human calcitonin (hCT) gene and express the gene in murine fibroblast NIH3T3 cells.


METHODS
A murine Igkappa-chain leader sequence and hCT gene were synthesized and cloned into pCDNA3.0 to form the pCDNA3.0-Igkappa-hCT eukaryotic expression vector, which was transfected into NIH3T3 cells. The mRNA and protein expressions and secretion of hCT were detected. Primarily cultured osteoclasts were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells, and their numbers were counted and morphology observed.


RESULTS
The expression and secretion of hCT were successfully detected in pCDNA3.0-Igk-hCT-transfected NIH3T3 cells. The number of osteoclasts was decreased and the cells became crumpled when they were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells.


CONCLUSIONS
A recombinant eukaryotic expression vector containing hCT gene was successfully constructed and expressed in NIH3T3 cells. The secreted recombinant hCT inhibited the growth and morphology of osteoclasts.
Li
Xiaolin
X
Department of Orthopedic Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, China. lixiaolin@sjtu.edu.cn
Jiang
Guozhong
G
Wu
Dan
D
Wang
Xiuli
X
Zeng
Bingfang
B
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002116:Calcitonin / Q000235:genetics* / Q000378:metabolism; D002469:Cell Separation; D018920:Coculture Techniques; D004274:DNA, Recombinant / Q000235:genetics*; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000201:enzymology; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase
D004274:DNA, Recombinant; D007527:Isoenzymes; D012333:RNA, Messenger; D002116:Calcitonin; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase
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