[Construction and identification of CerbB-2 siRNA expression plasmid and its transfer into human colon cancer cell lines HT-29].
OBJECTIVE
To construct a plasmid carrying small interfering RNA (siRNA) targeting human C-erbB2 gene (pGenesil- erbB2) and test its effect on Her-2 expression at the post-transcriptional level in human colon cancer cell lines HT-29 cells that highly express erbB2.


METHODS
A HT-29 cell line that highly expressed CerbB-2 was selected using immunohistochemical method. The double-stranded siRNA targeting human CerbB-2 cDNA and the negative control fragment were cloned into pGenesil-1 vector, and after identification and sequence analysis, the constructed pGenesil-erbB2 plasmid was transfected into the selected HT-29 cell line.


RESULTS
The pGenesil-erbB2 plasmid was successfully constructed and stably transferred into HT-29 cells. The transfection resulted in significant inhibition of Her-2 protein expression in the HT-29 cells, as shown by Western blotting.


CONCLUSIONS
The pGenesil-erbB plasmid we constructed can be stably transfected into HT-29 cells to inhibit the expression of Her-2 protein, and can be useful in further studies of increasing the radiosensitivity of HT-29 cell lines.
Center of Oncology, First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, China. zhaodongli@tom.com
Dong-li
DL
Zhao
Cheng-xue
CX
Dang
Yan-xia
YX
Sui
D001483:Base Sequence; D018734:Genes, erbB-2 / Q000235:genetics*; D019073:HT29 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human