Title: Construction of pEGFP-C1-miR-335 Recombinant Plasmid and Observation of its Expression in Breast Cancer Cells
Abstract: Objective To construct an Enhanced green fluorescent protein(EGFP)-labled euk-aryotic expression plasmid of microRNA gene with miR-335 and to detect its expression in breast cancer cell line MDA-MB-231. Methods With the technology of gene re-arrangement,miR-335 precursor sequence was amplified from the human genomic DNA,and was introduced into the restriction sites of pEGFP-C1.Then the correctness of the pEGFP-C1-miR-335 was evaluated by using restriction enzyme and sequencing analysis.The plasmid was transfected into the cell line MDA-MB-231 with lipofectin,the transient expression of GFP was observed under fluorescence microscope after 24 hours and detected by Northern blot. Results Correct construction of pEGFP-C1-miR-335 was identified by methods of restriction enzyme analysis and nucleotide sequence determination.A total of about 60% transfected cells emitted out green fluorescence under fluorescent microscope after 24 h of transfection.MiR-335 gene expressed by the transfected cells were testified by Northern blot. Conclusions The recombinant eukaryotic expression vectors have been constructed successfully and effectively expressed in breast cancer cells,which may provide an experimental basis for research of microRNA.
Publication Year: 2009
Publication Date: 2009-01-01
Language: en
Type: article
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