Construction and identification of a RNA interference plasmid for rat BNIP3 gene / 南方医科大学学报
Journal of Southern Medical University
;
(12): 2432-2435, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-323643
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The RNA interfering plasmid targeting rat BNIP3 has been constructed successfully, which provides a useful tool for studying the function of BNIP3.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
RNA, Messenger
/
Transfection
/
Proto-Oncogene Proteins
/
Mitochondrial Proteins
/
RNA, Small Interfering
/
HEK293 Cells
/
Genetic Vectors
/
Genetics
/
Membrane Proteins
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2010
Type:
Article
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