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Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
Published on: December 13, 2012
[Construction of an eukaryotic plasmid encoding HER2 and screening of a cell line stably expressing clones]
Teng-fei Xu1, Wen-qing Zhang, Hong Yu
1Pathogenic Biology Laboratory, Medical College, Qingdao University, Qingdao, China. tengfeixu@126.com
Aim:
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.
Conclusion:
An eukaryotic plasmid encoding HER2 (pcDNA6/v5-his-HER2) has been constructed and a cell line expressing HER2 stably has been prepared successfully.
