Recombinant plasmid pBG78 produces Sindbis virus (SINV) replicons in cells that can be easily visualized via GFP expression.
The Sindbis virus genome was inserted into the pcDNA3.1 Zeo+ expression vector such that the transcription start site of the CMV promoter overlaps with the first viral nucleotide of the viral genome. A Hepatitis D virus (HDV) ribozyme was added to the 3’ end of the viral genomic 35 base polyA sequence. The structural proteins from the 5’ subgenomic promoter were replaced with the Blasticidin-S-deaminase (BSD) gene for replicon selection. The enhanced green fluorescent protein (eGFP) gene was ligated into the unique XbaI restriction site after the 3’ subgenomic promoter to allow GFP to be produced during viral replication. Transfection of construct into mammalian cells produces a self-replicating Sindbis virus genome that expresses the BSD protein and GFP, that can be packaged into infectious virus-like particles by co-transfection with packaging plasmid pBG256 that can be used to insert the replicon into poorly or non-transfectable cells (in vitro or in vivo) for GFP expression.
From the laboratory of Brian J. Geiss, PhD, Colorado State University.
|Gene/insert name:||Sindbis virus replicon|
|Organism:||Sindbis Virus Strain TE3'2J (AR339)|
|Grow in E. coli at 37 C:||DH5-alpha or XL10-Gold E-coli at 37C|
|Cloning Site 5':||MluI|
|Cloning Site 3':||XhoI|
|Insert Size:||10,091 bp|
|Vector Backbone and Size:||pcDNA3.1 Zeo+|
|High or low copy:||High|
|Storage:||Room Temperature (Dried), -20C (liquid)|
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