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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

Efficiency of recombining genes into pBG60 vector. A. Bacterial colonies obtained from recombination reactions. B. Structure of the pBG78 2nd SGP and position of asymmetric colony PCR primer pair. C. Colony PCR of 14 independent clones from colonies in Figure 2A. D. pBG78 plasmids produce replication-competent RNA in BHK cells. BHK cells were transfected with one random pBG78 clone and fluorescence signal was detected 48 hours post transfection.
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Figure 2: Efficiency of recombining genes into pBG60 vector. A. Bacterial colonies obtained from recombination reactions. B. Structure of the pBG78 2nd SGP and position of asymmetric colony PCR primer pair. C. Colony PCR of 14 independent clones from colonies in Figure 2A. D. pBG78 plasmids produce replication-competent RNA in BHK cells. BHK cells were transfected with one random pBG78 clone and fluorescence signal was detected 48 hours post transfection.

Mentions: We first tested the ability of the replicon plasmid pBG60 (Figure 1) to incorporate an attL flanked insert in a LR recombination reaction. Recombination of the attR-containing replicon plasmid (pBG60) with an attL flanked GFP entry plasmid (pBG76) in a LR Clonase II reaction resulted in a large number of colonies when transformed into DH5α cells (Figure 2A), whereas transformation with pBG76 or pBG60 incubated with LR Clonase resulted in almost no colonies. Fourteen colonies from the pBG60–pBG76 recombination (referred to as pBG78, Figure 1) were examined for insert size and orientation by asymmetric colony PCR with primers BG192/BG209 (Figure 2B). All of the colonies produced a PCR product of 1 Kb, indicating that the GFP gene was present in the replicon plasmid in the correct orientation. Sequencing the insert region of several of the plasmids verified the presence and orientation of the insert, and the reconstitution of the attB1 and attB2 sites flanking the GFP open reading frame. A related replicon plasmid which contains the attR recombination cassette in the attR2/attR1 orientation with respect to the second SGP (pBG59), was similarly able to incorporate attL flanked inserts, but in reverse orientation (data not shown). Finally, transfection of the one of the pBG78 plasmid clones into BHK cells results in GFP expression 2 days after transfection (Figure 2C). Because the expression of genes downstream of the 3' SGP are dependent on replicon RNA replication, the expression of GFP indicates that the replicon is actively replicating and that the GFP gene is being expressed in a replicon dependent manner. Therefore, the presence of the attB recombination sites does not interfere with expression of the inserted gene from the SGP or replication of the replicon RNA.


Recombination-ready Sindbis replicon expression vectors for transgene expression.

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

Efficiency of recombining genes into pBG60 vector. A. Bacterial colonies obtained from recombination reactions. B. Structure of the pBG78 2nd SGP and position of asymmetric colony PCR primer pair. C. Colony PCR of 14 independent clones from colonies in Figure 2A. D. pBG78 plasmids produce replication-competent RNA in BHK cells. BHK cells were transfected with one random pBG78 clone and fluorescence signal was detected 48 hours post transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Efficiency of recombining genes into pBG60 vector. A. Bacterial colonies obtained from recombination reactions. B. Structure of the pBG78 2nd SGP and position of asymmetric colony PCR primer pair. C. Colony PCR of 14 independent clones from colonies in Figure 2A. D. pBG78 plasmids produce replication-competent RNA in BHK cells. BHK cells were transfected with one random pBG78 clone and fluorescence signal was detected 48 hours post transfection.
Mentions: We first tested the ability of the replicon plasmid pBG60 (Figure 1) to incorporate an attL flanked insert in a LR recombination reaction. Recombination of the attR-containing replicon plasmid (pBG60) with an attL flanked GFP entry plasmid (pBG76) in a LR Clonase II reaction resulted in a large number of colonies when transformed into DH5α cells (Figure 2A), whereas transformation with pBG76 or pBG60 incubated with LR Clonase resulted in almost no colonies. Fourteen colonies from the pBG60–pBG76 recombination (referred to as pBG78, Figure 1) were examined for insert size and orientation by asymmetric colony PCR with primers BG192/BG209 (Figure 2B). All of the colonies produced a PCR product of 1 Kb, indicating that the GFP gene was present in the replicon plasmid in the correct orientation. Sequencing the insert region of several of the plasmids verified the presence and orientation of the insert, and the reconstitution of the attB1 and attB2 sites flanking the GFP open reading frame. A related replicon plasmid which contains the attR recombination cassette in the attR2/attR1 orientation with respect to the second SGP (pBG59), was similarly able to incorporate attL flanked inserts, but in reverse orientation (data not shown). Finally, transfection of the one of the pBG78 plasmid clones into BHK cells results in GFP expression 2 days after transfection (Figure 2C). Because the expression of genes downstream of the 3' SGP are dependent on replicon RNA replication, the expression of GFP indicates that the replicon is actively replicating and that the GFP gene is being expressed in a replicon dependent manner. Therefore, the presence of the attB recombination sites does not interfere with expression of the inserted gene from the SGP or replication of the replicon RNA.

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