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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

Packaging and infection of pBG78 replicons in BHK and C6/36 cells. pBG78 PIPS were generated by co-transfecting pBG78 and pBG44 plasmid DNA into BHK cells. Media was collected 24 hrs post transfection and 100 ul of the clarified media was added to new BHK or C6/36 cells. 24 hrs after infection the media was replaced with fresh media containing10 ug/ml blasticidin and the cultures were maintained for an additional three weeks. Media was replaced every 3–4 days. GFP fluorescence was detected at 3 days post infection (panels B and E) and 3 weeks post infection (panels D and H).
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Figure 4: Packaging and infection of pBG78 replicons in BHK and C6/36 cells. pBG78 PIPS were generated by co-transfecting pBG78 and pBG44 plasmid DNA into BHK cells. Media was collected 24 hrs post transfection and 100 ul of the clarified media was added to new BHK or C6/36 cells. 24 hrs after infection the media was replaced with fresh media containing10 ug/ml blasticidin and the cultures were maintained for an additional three weeks. Media was replaced every 3–4 days. GFP fluorescence was detected at 3 days post infection (panels B and E) and 3 weeks post infection (panels D and H).

Mentions: The ability of the replicon RNA to be packaged into PIPs and infect naive cells was tested. BHK cells were transfected with GFP expressing replicon plasmid pBG78 either alone or with the packaging plasmid pBG44. Transfection of pBG78 alone resulted in ~20% of the cells expressing GFP and the remaining cells were GFP negative (Figure 4, Panel b). However, co-transfection of pBG78 and pBG44 resulted in almost all the cells in the cultures expressing GFP either at high or medium levels (Figure 4, Panel d). This suggests that the transfected cells are producing PIPs that are spreading through the culture to infect remaining naive cells. We next tested if we could use PIPs to transfer the replicons to mosquito C6/36 cells. Supernatant from BHK cells transfected with pBG78 and pBG44 were collected, clarified by centrifugation, and added to C6/36 cell cultures. The infected C6/36 cells were treated with blasticidin two days after infection, and were cultured from an additional 3 weeks in the presence of drug. GFP fluorescence was detected in adjacent cells 3 days after infection, and large colonies of GFP expressing cells were seen 3 weeks after infection (Figure 4, panels E and H). The number of GFP positive cells from the initial infection was relatively low following media transfer, but the formation of blasticidin resistant, GFP expressing colonies indicates that the replicon RNA can be transferred to naive insect cells and establish persistent infection.


Recombination-ready Sindbis replicon expression vectors for transgene expression.

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

Packaging and infection of pBG78 replicons in BHK and C6/36 cells. pBG78 PIPS were generated by co-transfecting pBG78 and pBG44 plasmid DNA into BHK cells. Media was collected 24 hrs post transfection and 100 ul of the clarified media was added to new BHK or C6/36 cells. 24 hrs after infection the media was replaced with fresh media containing10 ug/ml blasticidin and the cultures were maintained for an additional three weeks. Media was replaced every 3–4 days. GFP fluorescence was detected at 3 days post infection (panels B and E) and 3 weeks post infection (panels D and H).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Packaging and infection of pBG78 replicons in BHK and C6/36 cells. pBG78 PIPS were generated by co-transfecting pBG78 and pBG44 plasmid DNA into BHK cells. Media was collected 24 hrs post transfection and 100 ul of the clarified media was added to new BHK or C6/36 cells. 24 hrs after infection the media was replaced with fresh media containing10 ug/ml blasticidin and the cultures were maintained for an additional three weeks. Media was replaced every 3–4 days. GFP fluorescence was detected at 3 days post infection (panels B and E) and 3 weeks post infection (panels D and H).
Mentions: The ability of the replicon RNA to be packaged into PIPs and infect naive cells was tested. BHK cells were transfected with GFP expressing replicon plasmid pBG78 either alone or with the packaging plasmid pBG44. Transfection of pBG78 alone resulted in ~20% of the cells expressing GFP and the remaining cells were GFP negative (Figure 4, Panel b). However, co-transfection of pBG78 and pBG44 resulted in almost all the cells in the cultures expressing GFP either at high or medium levels (Figure 4, Panel d). This suggests that the transfected cells are producing PIPs that are spreading through the culture to infect remaining naive cells. We next tested if we could use PIPs to transfer the replicons to mosquito C6/36 cells. Supernatant from BHK cells transfected with pBG78 and pBG44 were collected, clarified by centrifugation, and added to C6/36 cell cultures. The infected C6/36 cells were treated with blasticidin two days after infection, and were cultured from an additional 3 weeks in the presence of drug. GFP fluorescence was detected in adjacent cells 3 days after infection, and large colonies of GFP expressing cells were seen 3 weeks after infection (Figure 4, panels E and H). The number of GFP positive cells from the initial infection was relatively low following media transfer, but the formation of blasticidin resistant, GFP expressing colonies indicates that the replicon RNA can be transferred to naive insect cells and establish persistent infection.

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