Author
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Estevez, Carlos |
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Yu, Qingzhong |
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Submitted to: Southeastern Regional Virology Conference
Publication Type: Abstract Only Publication Acceptance Date: 2/10/2006 Publication Date: 3/17/2006 Citation: Estevez, C., Yu, Q. 2006. Development of a green fluorescent protein (gfp)-based minireplicon system for newcastle disease virus [abstract]. Ninth Southeastern Regional Virology Conference Program Book. p. 15. Interpretive Summary: Technical Abstract: Minireplicon systems are helpful tools for analyses of cis and trans-acting elements required for gene expression of negative stranded RNA viruses. In the case of Newcastle disease virus (NDV), most minireplicon systems have been developed using the Chloramphenicol Acetyl Transferase (CAT) as a reporter. The detection of the CAT expression requires lysis of transfected cell monolayers, detection of activity by chemical substrates and determination of the intensity of the reactions by means of spectrophotometry or thin layer chromatography. The present work describes the development of a NDV minireplicon system which is composed of four plasmids: one coding for the GFP gene flanked by the NDV leader and trailer sequences; and three protein expression plasmids coding for the nucleocapsid (N), phosphoprotein (P) and polymerase (L) genes of the virus. Expression of the GFP reporter gene was assessed by direct visualization of fluorescence in the transfected cell monolayers, without the need for cell lysis or any chemical reactions. This minireplicon system is highly specific as evidenced by the complete absence of fluorescence in the cell monolayers transfected with all but the L gene coding plasmid, which were used as negative controls. Furthermore, this system allows for analysis of the differences of GFP expression when mutagenesis studies of cis or trans-acting elements are performed. |
