[Construction of an eukaryotic plasmid encoding HER2 and screening of a cell line stably expressing clones].
OBJECTIVE
To construct an eukaryotic vector encoding extracellular domain of human epidermal growth factor receptors (HER2), pcDNA6/v5-his-HER2, and to screen HER2 positive clones from mouse breast cancer cell line EMT6.


METHODS
The extracellular domain of HER2 was amplified from pcDNA3.1-HER2 by PCR. pcDNA6/v5-his-HER2 was prepared by inserting the fragment into the plasmid pcDNA6/v5-his. Then the recombinant vector was identified by restriction enzyme and sequencing. Next, pcDNA6/v5-his-HER2 was transfected into the EMT6 cell line and the positive clones (EMT6/HER2) were screened with blasticidin. Finally, the expression of HER2 in EMT6/HER2 was detected by RT-PCR and immunohistochemistry.


RESULTS
The fragment of HER2 was amplified and pcDNA6/v5-his-HER2 was prepared successfully. No errors were found both in the sequence and ORF of the acquired fragment. The expected fragment of HER2 (1896 bp) was amplified from EMT6/HER2 by RT-PCR and positive signals of HER2 were detected in EMT6/HER2 by immunohistochemistry.


CONCLUSIONS
An eukaryotic plasmid encoding HER2 (pcDNA6/v5-his-HER2) has been constructed and a cell line expressing HER2 stably has been prepared successfully.
Xu
Teng-fei
TF
Pathogenic Biology Laboratory, Medical College, Qingdao University, Qingdao, China. tengfeixu@126.com
Zhang
Wen-qing
WQ
Yu
Hong
H
Li
Dan
D
D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D009705:Nucleosides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D009705:Nucleosides; D018719:Erb-b2 Receptor Tyrosine Kinases; C004500:blasticidin S