Recombination-ready Sindbis replicon expression vectors for transgene expression.
BACKGROUND
Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation.


RESULTS
Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis.


CONCLUSIONS
Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.
Geiss
Brian J
BJ
Arthropod-Borne and Infectious Diseases Laboratory, Department of Molecular Biology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA. Brian.Geiss@colostate.edu
Shimonkevitz
Lisa H
LH
Sackal
Cherilyn I
CI
Olson
Ken E
KE
D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D011995:Recombination, Genetic; D012093:Replicon; D012845:Sindbis Virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D012367:RNA, Viral