The Sindbis virus genome lacking the viral nsP1-nsP4 replication genes and possessing the viral structural protein genes under the control of the native viral subgenomic promoter 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. Transfection this construct into mammalian cells produces a Sindbis virus genome that cannot express the viral structural proteins unless the viral nsP1-nsP4 replication proteins are provided in trans by a co-transfected replicon plasmid (pBG60, pBG68, pBG78). This construct was found to be superior to the pBG44 packaging construct described in Geiss et al., 2007 in production of virus-like particles. Equimolar amounts of replicon and packaging plasmids should be transfected into cells for optimal results.
From the laboratory of Brian J. Geiss, PhD, Colorado State University.
|Gene/insert name:||Sindbis virus replicon packaging plasmid|
|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:||5,476 bp|
|Vector Backbone and Size:||pcDNA3.1 Zeo+|
|High or low copy:||High|
|Storage:||Room Temperature (Dried), -20C (liquid)|
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