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Recombination-ready Sindbis replicon expression vectors for transgene expression.

Geiss BJ, Shimonkevitz LH, Sackal CI, Olson KE - Virol. J. (2007)

Bottom Line: Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Conclusion: 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.

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Related in: MedlinePlus

SINV replicons can express epitope tagged mosquito genes for several days. A. C6/36, S2, or BHK cells were infected with PIPs expressing a V5 epitope-tagged Ae. aegypti R2D2 cDNA either in a sense (pBG156) or antisense (pBG155) orientation. Cells were treated with blasticidin for four days and V5-R2D2 detected by western blot analysis. B. C6/36 cells were co-cultured with pBG156/pBG44 co-transfected BHK cells, transferred to new plates, and samples of cells collected at the indicated times for western blot analysis.
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Figure 6: SINV replicons can express epitope tagged mosquito genes for several days. A. C6/36, S2, or BHK cells were infected with PIPs expressing a V5 epitope-tagged Ae. aegypti R2D2 cDNA either in a sense (pBG156) or antisense (pBG155) orientation. Cells were treated with blasticidin for four days and V5-R2D2 detected by western blot analysis. B. C6/36 cells were co-cultured with pBG156/pBG44 co-transfected BHK cells, transferred to new plates, and samples of cells collected at the indicated times for western blot analysis.

Mentions: We used our co-culture protocol to express an epitope-tagged version of an endogenous Aedes aegypti RNAi component, R2D2. C6/36 or S2 cells were co-culture infected with PIPs produced from the R2D2 expressing replicon plasmid pBG156 or pBG155 (R2D2 antisense), and selected with blasticidin. Four days after blasticidin treatment 1 × 105 cells were collected, lysed, and the protein resolved on SDS-PAGE. Western blot analysis of the V5 epitope-tagged R2D2 protein showed high levels of expression in C6/36 cells (Figure 6A, lane 2). However, we were unable to detect any Ae. aegypti R2D2 protein in infected S2 cells (Figure 6A, lane 4). Drosophila R2D2 has been previously shown to be unstable in the absence of DCR2 [19], so these results may represent the inability of Drosophila DCR2 to stabilize Ae. aegypti R2D2. Interestingly, R2D2 is stable in the BHK cells used in for the co-culture experiments (Figure 6A, lane 5). We then tested if replicon-expressed R2D2 can be stably expressed in C6/36 cells for multiple days following infection. We were able to detect R2D2 expression for at least 4 days post co-culture without blasticidin treatment (Figure 6B), indicating either that the protein is very stable in C6/36 cells or that the replicon can express the protein for several days post-infection. These experiments indicate that double-subgenomic replicons can be used to express and study the biology of individual genes in insect cells in a rapid manner.


Recombination-ready Sindbis replicon expression vectors for transgene expression.

Geiss BJ, Shimonkevitz LH, Sackal CI, Olson KE - Virol. J. (2007)

SINV replicons can express epitope tagged mosquito genes for several days. A. C6/36, S2, or BHK cells were infected with PIPs expressing a V5 epitope-tagged Ae. aegypti R2D2 cDNA either in a sense (pBG156) or antisense (pBG155) orientation. Cells were treated with blasticidin for four days and V5-R2D2 detected by western blot analysis. B. C6/36 cells were co-cultured with pBG156/pBG44 co-transfected BHK cells, transferred to new plates, and samples of cells collected at the indicated times for western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2164957&req=5

Figure 6: SINV replicons can express epitope tagged mosquito genes for several days. A. C6/36, S2, or BHK cells were infected with PIPs expressing a V5 epitope-tagged Ae. aegypti R2D2 cDNA either in a sense (pBG156) or antisense (pBG155) orientation. Cells were treated with blasticidin for four days and V5-R2D2 detected by western blot analysis. B. C6/36 cells were co-cultured with pBG156/pBG44 co-transfected BHK cells, transferred to new plates, and samples of cells collected at the indicated times for western blot analysis.
Mentions: We used our co-culture protocol to express an epitope-tagged version of an endogenous Aedes aegypti RNAi component, R2D2. C6/36 or S2 cells were co-culture infected with PIPs produced from the R2D2 expressing replicon plasmid pBG156 or pBG155 (R2D2 antisense), and selected with blasticidin. Four days after blasticidin treatment 1 × 105 cells were collected, lysed, and the protein resolved on SDS-PAGE. Western blot analysis of the V5 epitope-tagged R2D2 protein showed high levels of expression in C6/36 cells (Figure 6A, lane 2). However, we were unable to detect any Ae. aegypti R2D2 protein in infected S2 cells (Figure 6A, lane 4). Drosophila R2D2 has been previously shown to be unstable in the absence of DCR2 [19], so these results may represent the inability of Drosophila DCR2 to stabilize Ae. aegypti R2D2. Interestingly, R2D2 is stable in the BHK cells used in for the co-culture experiments (Figure 6A, lane 5). We then tested if replicon-expressed R2D2 can be stably expressed in C6/36 cells for multiple days following infection. We were able to detect R2D2 expression for at least 4 days post co-culture without blasticidin treatment (Figure 6B), indicating either that the protein is very stable in C6/36 cells or that the replicon can express the protein for several days post-infection. These experiments indicate that double-subgenomic replicons can be used to express and study the biology of individual genes in insect cells in a rapid manner.

Bottom Line: Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus