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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

Structure of the Gateway SINV replicon plasmids pBG60 and pBG78. Transcription of the viral RNA is initiated by the cytomegalovirus immediate early (CMV) promoter and terminated by a polyadenylation signal 3' of the HDV ribozyme (not shown). The hepatitis delta virus (HDV) ribozyme trans-cleaves the RNA, resulting in an authentic 3' RNA end. bsd represents the blasticidin S-deaminase gene. The Gateway attR1/attR2 recombination sites are positioned 3' of the 2nd SGP. CcdB represents E. coli DNA Gyrase poison. Cm(R) represents chloramphenicol acetyltransferase. Recombination of the pBG60 plasmid with pBG76 results in the pBG78 plasmid.
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Figure 1: Structure of the Gateway SINV replicon plasmids pBG60 and pBG78. Transcription of the viral RNA is initiated by the cytomegalovirus immediate early (CMV) promoter and terminated by a polyadenylation signal 3' of the HDV ribozyme (not shown). The hepatitis delta virus (HDV) ribozyme trans-cleaves the RNA, resulting in an authentic 3' RNA end. bsd represents the blasticidin S-deaminase gene. The Gateway attR1/attR2 recombination sites are positioned 3' of the 2nd SGP. CcdB represents E. coli DNA Gyrase poison. Cm(R) represents chloramphenicol acetyltransferase. Recombination of the pBG60 plasmid with pBG76 results in the pBG78 plasmid.

Mentions: The attR1/attR2 recombination cassette was PCR amplified from Gateway pDEST32 (Invitrogen) with primers BG121 and BG122. BG121 and BG122 contain NheI restriction sites, and were used to ligate the recombination cassette into XbaI digested pBG68. pBG60 contains the recombination cassette in the attR1/attR2 orientation with respect to the second SGP. pBG60 was propagated in the E. coli strain DB3.1, which is resistant to the CcdB gene present in the attR recombination cassette. pBG60 (Figure 1) is considered a destination plasmid competent for recombination with attL1/attL2 flanked DNA sequences.


Recombination-ready Sindbis replicon expression vectors for transgene expression.

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

Structure of the Gateway SINV replicon plasmids pBG60 and pBG78. Transcription of the viral RNA is initiated by the cytomegalovirus immediate early (CMV) promoter and terminated by a polyadenylation signal 3' of the HDV ribozyme (not shown). The hepatitis delta virus (HDV) ribozyme trans-cleaves the RNA, resulting in an authentic 3' RNA end. bsd represents the blasticidin S-deaminase gene. The Gateway attR1/attR2 recombination sites are positioned 3' of the 2nd SGP. CcdB represents E. coli DNA Gyrase poison. Cm(R) represents chloramphenicol acetyltransferase. Recombination of the pBG60 plasmid with pBG76 results in the pBG78 plasmid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Structure of the Gateway SINV replicon plasmids pBG60 and pBG78. Transcription of the viral RNA is initiated by the cytomegalovirus immediate early (CMV) promoter and terminated by a polyadenylation signal 3' of the HDV ribozyme (not shown). The hepatitis delta virus (HDV) ribozyme trans-cleaves the RNA, resulting in an authentic 3' RNA end. bsd represents the blasticidin S-deaminase gene. The Gateway attR1/attR2 recombination sites are positioned 3' of the 2nd SGP. CcdB represents E. coli DNA Gyrase poison. Cm(R) represents chloramphenicol acetyltransferase. Recombination of the pBG60 plasmid with pBG76 results in the pBG78 plasmid.
Mentions: The attR1/attR2 recombination cassette was PCR amplified from Gateway pDEST32 (Invitrogen) with primers BG121 and BG122. BG121 and BG122 contain NheI restriction sites, and were used to ligate the recombination cassette into XbaI digested pBG68. pBG60 contains the recombination cassette in the attR1/attR2 orientation with respect to the second SGP. pBG60 was propagated in the E. coli strain DB3.1, which is resistant to the CcdB gene present in the attR recombination cassette. pBG60 (Figure 1) is considered a destination plasmid competent for recombination with attL1/attL2 flanked DNA sequences.

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