title: [Construction and identification of a RNA interference plasmid for rat BNIP3 gene].
abstract: OBJECTIVE
  To construct a RNA interfering plasmid targeting rat Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and assess its effect on exogenous BNIP3 expression in HEK293 cells.
  
  
  METHODS
  The miRNA sequences were designed using Invitrogen BLOCK-iT RNAi Designer and synthesized into double-strand oligonucleotides, which were cloned into the pcDNATM6.2-GW/EmGFP-miR vector, followed by transformation of the product into competent Top10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced before its co-transfection with pEGFP-C3- rBNIP3 plasmid into HEK293 cells. The interference effect of the constructed plasmid on BNIP3 mRNA and protein expression were detected by real-time PCR and Western blotting.
  
  
  RESULTS
  The sequencing result indicated that the interfering plasmid targeting rat BNIP3 was constructed correctly. After transfection into HEK293 cells, the interfering plasmid significantly inhibited exogenous BNIP3 mRNA and protein expressions.
  
  
  CONCLUSIONS
  The RNA interfering plasmid targeting rat BNIP3 has been constructed successfully, which provides a useful tool for studying the function of BNIP3.
authors.affiliation: Department of Neurobiology, College of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China. shycm@fimmu.com
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.forename: Xing-mei
authors.initials: XM
authors.lastname: Zhang
authors.forename: Bo-xing
authors.initials: BX
authors.lastname: Li
authors.forename: Hong-yu
authors.initials: HY
authors.lastname: Sun
authors.forename: Xiao-wen
authors.initials: XW
authors.lastname: Li
authors.forename: Tian-ming
authors.initials: TM
authors.lastname: Gao
mesh_terms: D000818:Animals; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D024101:Mitochondrial Proteins; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D014162:Transfection
chemical_list: C505650:BNIP3 protein, rat; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering