Recombination-ready Sindbis replicon expression vectors for transgene expression
© Geiss et al; licensee BioMed Central Ltd. 2007
Received: 18 September 2007
Accepted: 26 October 2007
Published: 26 October 2007
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.
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.
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.
Alphaviruses, such as Sindbis virus (SINV), are positive-sense RNA viruses that have been extensively used for transgene expression in mammalian and insect systems. SINV are ideal for transgene expression because they can express high levels of exogenous protein or RNA [1, 2], infect a wide range of species [1, 3], stably replicate for long periods of time with minimal cytotoxicity [1, 3, 4], and do not integrate into the host genome. Two main approaches have been used for SINV transduction systems: 1) non-infectious subgenomic replicons that express exogenous genes from the native subgenomic promoter (SGP) that normally would express the structural proteins, and 2) infectious SINV containing an engineered second SGP for gene expression. Subgenomic replicons are capable of authentic viral RNA replication and can express exogenous genes, but are unable to form viral particles capable of intercellular spread because they lack structural proteins that form virus particles. The lack of intercellular spread by the replicons allows for the isolation of clonal cell populations that express a gene of interest, which is useful if the replicon is being used to generate a homogenous population of cells. SINV replicons have been used in a wide range of applications, including expression of reporter genes , gene therapy , vaccination , and expression of heterologous viral proteins . The addition of a drug resistance gene to a replicon, such as blasticidin S-deaminase (bsd) or puromycin N-acetyltransferase , can result in long term persistent infection and selection for cells containing replicons. Because the SGP is small and well defined, SINV replicons containing multiple SGPs have been generated that allow for simultaneous drug selection and transgene expression . Infectious SINV containing a second SGP have recently been used to express green fluorescent protein in vivo, allowing real-time visualization of viral infection in mosquito vectors [9, 10]. Co-transfecting replicon containing cells with a plasmid that expresses the viral structural proteins can produce pseudo-infectious viral particles (PIPs) that can initiate a single round of infection but are unable to spread to additional cells. PIPs are useful because they can be generated at high titers and have the same tropism as infectious SINV but are not able to spread from the cell they infect. Thus SINV replicons are a valuable tool for transgene expression both in cell culture and in live animals.
While SINV replicons are very useful for studying gene expression, they historically have had several drawbacks that make them difficult to use. One major problem is generating and delivering replication competent replicon RNA into target cells. Replicon RNA has classically been generated by in vitro transcription in the presence of nucleotide cap analog, and the resulting RNA is electroporated into the cells. Transcription and RNA electroporation require specialized protocols and equipment that are not readily available in all laboratories. Replicon expression plasmids that use mammalian promoters to transcribe replicon RNA from transfected plasmids have been developed to circumvent these issues . Expression plasmids allow replicons to be generated simply by transfecting plasmid DNA into mammalian cells, and the replicon RNA can be packaged into PIPs by co-transfection with a separate packaging plasmid or transfection into a packaging cell line .
A second issue is engineering the insert of interest into the replicon plasmid in the proper orientation. DNA coding for genes is ligated into the replicon plasmid using a unique restriction site 3' to the SGP, allowing virus-mediated transcription of the insert DNA to occur . The insert DNA can be ligated into the replicon plasmid in either a sense or antisense orientation, resulting in a mixed population of recombinants that must be screened for clones of the appropriate orientation. Screening individual clones for orientation is not a difficult task if only one clone is desired, but the inability to guide insert orientation or select for recombinant replicon plasmids makes the development of replicon libraries difficult because inserts will be present in both orientations or in multiple copies. Additionally, if the restriction site used is found within the insert DNA sequence, alternate cloning strategies must be employed to avoid truncating the insert. If a library is being constructed, the complexity of the usable library will likely be reduced because many cDNAs will contain the restriction site. To address the problem of cloning inserts into SINV replicons efficiently and in the correct orientation, we have constructed a replicon expression plasmid that contain lambda phage attR Gateway™ recombination sequences 3' of the second SGP. The attR recombination sequences flank a negative selection marker that increases the rate of correct recombination and reduces the number of non-recombinant clones to be screened. The attR1 and attR2 recombination sites, which allow specific recombination between attL1 and attL2 sites in the presence of LR Clonase enzyme mixture, are positioned in either a sense or antisense orientation, allowing highly specific and directional recombination between the replicon plasmids and attL1/attL2 flanked insert sequences. The experiments described show that addition of recombination technology increases the speed and efficiency of replicon plasmid construction, make the manipulation of alphavirus replicons easier and more user-friendly to the non-expert user, and provides a foundation for the establishment of SINV replicon-mediated expression cloning protocols.
Materials and methods
Construction of replicon expression plasmids, donor plasmids, and packaging plasmid
5' GGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGATTGACGG CGTAGTACACACTATTGAATCAAACAGCCG
5' CGGCTGTTTGATTCAATAGTGTGTACTACGCC GTCAATCGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCC
5' GGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAATGTTAAAAACAAAATTTTG
5' ATTACCGAGGGGACGGTCCCCTCGGAATGTTGCCCAGCCG GCGCCAGCGAGGAGGCTGGGACCATGCCGGCC
5' GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGGGATGCA TGGTACCATGGTGAGCAAGG
5' GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGCCTCAAAG CCAGTCCTGAGCACG
Donor plasmid pBG76 contains the green fluorescent protein gene flanked by attL1 and attL2 recombination sites. To construct pBG76, primers BG155 and BG156, which contain attB1 and attB2 recombination sites were used to PCR amplify eGFP from pIE-GFP (Clontech). The PCR product was incubated in a BP Clonase recombination reaction with entry plasmid pDONR222 (Invitrogen) for 1 hr at 25°C. The reaction was electroporated into XL-1 Blue E. coli cells and plated onto kanamycin-containing agar plates. Donor plasmid pBG151, which encodes a V5 epitope-tagged Ae. aegypti R2D2 cDNA flanked by attL1/attL2 sites, was constructed in a similar manner to pBG76 using primers BG256/BG258.
pBG44, which expresses the SINV structural proteins from a CMV promoter, was constructed by PCR amplifying the structural protein open reading frame from pTE/3'2J with primers BG115 and BG116. The PCR product was digested with HindIII and ApaI, gel purified, and ligated into the pcDNA 3.1 Zeo (+) using the same restriction sites.
All in silico DNA manipulation was performed with Vector NTI 10 Suite (Invitrogen).
Recombination ready C6/36 cDNA library preparation and recombination reactions
cDNA libraries were constructed using the CloneMiner cDNA Construction Kit (Invitrogen) as recommended by the manufacturer. A representative cDNA library was prepared from actively growing C6/36 (Aedes albopictus) cells. Briefly, mRNA was isolated from 1.0 × 107 C6/36 cells using the Micro-FastTrack 2.0 mRNA Isolation kit (Invitrogen). cDNAs were synthesized with attB1 recombination sites at the 5' terminus of the cDNA and attB2 recombination sites at the 3' poly-A tail of the cDNA. Isolated cDNAs were recombined using BP Clonase into the pDONR222 plasmid, electroporated into DH10B E. coli cells, and aliquots stored as glycerol stocks at -80°C. The size of the library was determined in colony forming units/ml of glycerol stock from an individual aliquot. Multiple clones were sequenced to verify appropriate recombination. A portion of the cDNA library plasmid containing larger inserts was isolated by agarose gel extraction.
A completely defined micro-scale cDNA library containing inserts of greater that 1 Kb was generated from gel extracted plasmids derived from the C6/36 cDNA library described above. The gel-extracted plasmids were transformed into DH5α cells. Ten individual clones were isolated and the size of the insert regions was determined by BsrGI restriction enzyme digestion. The micro-scale cDNA library was generated by mixing equal molar amounts of each of the ten clones, and the micro-scale cDNA library was used in a LR Clonase reaction with pBG60. The product of the recombination reaction was transformed into DH5α cells (CcdB sensitive) and analyzed for insert size by colony PCR and BsrGI restriction digestion.
Cell culture, transfection, and replicon packaging
Baby hamster kidney (BHK) cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS) as previously described . BHK cells were transfected using Lipofectamine 2000 following the manufacturer's instructions. C6/36 cells were cultured in L-15 insect medium supplemented with 10% FBS as previously described . S2 cells were cultured is Schneider's media containing 10% FBS. Replicon-containing C6/36 cells were maintained in the presence of 6 ug/ml blasticidin (Invitrogen) for at least 2 weeks prior to assay, and replicon-containing S2 cells were maintained in the presence of 10 ug/ml blasticidin for 4 days prior to assay. C6/36 and S2 cells were transfected using Insect Gene Juice transfection reagent (EMD Biosciences), using a ratio of 10 μl reagent/μg DNA.
To generate PIPs, replicon expression plasmids were co-transfected with the packaging plasmid pBG44 into subconfluent BHK cells. Cells were washed extensively 4 hours after transfection to remove remaining plasmid DNA and transfection reagent. Twenty-four hours later media from the transfected BHK cells was collected, centrifuged at 13,000 × G to remove any cell debris, and the clarified supernatant was collected and frozen at -80°C. To infect C6/36 cells, the supernatant was added directly to subconfluent C6/36 cells. Twenty-four hours after infection, the media was replaced with fresh complete L-15 media containing 10 μg/ml blasticidin and the cells were selected for an additional 2 weeks. Conditions for co-culture assays are described in the results section.
GFP expression in live BHK and C6/36 cells was visualized on an Olympus inverted fluorescence microscope and images were captured with a coupled CCD camera.
Western blot analysis
To prepare cell samples, equal numbers of C6/36 cells were boiled in 1× SDS loading buffer and clarified by centrifugation at 13,000 × G. Equal volumes were loaded and resolved on 12% SDS-PAGE gels, and proteins were transferred to nitrocellulose membranes. Membranes were incubated Anti-V5 antibody (Invitrogen), then incubated with horse radish peroxidase-conjugated anti-mouse secondary antibody. Proteins were detected with ECL Plus Western Blot Detection kit (Amersham) on a Storm 860 phosphorimager.
Colony PCR and reverse transcription of replicon insert regions
Colony PCR was used to determine the size of inserts recombined into the pBG60 plasmid. Individual colonies from transformed BP or LR reactions were touched with a 10 μl pipette tip and mixed into a 20 μl PCR reaction containing 1 pM primers, recombinant Taq (Invitrogen), 0.2 mM dNTP, and 5% DMSO. 1 μl of the bacteria-containing colony PCR reaction was spotted onto ampicillin-containing agar plates prior to thermocycling for archiving. Colony PCR reactions were subjected to 40 cycles of (95°C (30 s)/55°C (30 s)/72°C (1 m/kb)) and products were resolved on 1% agarose gels.
RNA from replicon-containing C6/36 cells was isolated using RNeasy RNA isolation kits (Qiagen). One microgram of total RNA was reverse transcribed with replicon-specific primers BG162/BG192 using the Superscript One-Step RT PCR system (Invitrogen). The resulting PCR product was purified using a Qiaquick PCR prep kit (Qiagen), and the PCR product was recombined into pDONR222 in BP clonase reactions. Recombination reactions were transformed into DH5α E. coli cells and grown on 50 mg/ml kanamycin plates. Individual colonies were tested for inserts by colony PCR, and select clones were verified by sequencing.
Replicon insert recombination is efficient and directional
attL-flanked cDNAs of varying sizes can be efficiently recombined into replicon plasmids
Replicons can be packaged into PIPs and used to infect naive mammalian or insect cells
Insert regions from persistently replicating replicons can be rapidly isolated and recombined into entry plasmids
In this report we describe the construction and testing of a new recombination-based system for manipulating expression cassettes in Sindbis virus replicons and show their utility for expressing transgenes of interest in cultured mosquito cells. The replicon expressing plasmids we have developed have several features that make them unique, including replicon-expressed blasticidin drug resistance and the ability to specifically and efficiently insert a transgene of interest 3' to the second SGP by directional recombination.
The inclusion of the recombination cassettes into replicon expression plasmids make Sindbis replicons much easier to use compared to previously generations replicon expression plasmids. Because exogenous genes or DNA sequences can be inserted into the replicon by recombination rather than using restriction sites, it is much more likely that full-length genes can be inserted into the replicon. Such a feature becomes important if the replicon is to be used to express full-length genes, because large cDNAs are more likely to contain restriction sites that would make cloning the desired insert into the replicon more difficult. Additionally, recombination is directional and efficient. Almost all of the LR recombined replicon plasmids we tested contained the desired insert in the correct orientation. Finally, recombination is rapid. Once the DNA of interest has been recombined into an Entry plasmid, recombination of the attL flanked DNA into the attR containing replicons takes only 1 hour, in contrast to the many hours necessary for generating a replicon by restriction digestion and ligation.
There are several potential applications for recombination-compatible replicon plasmids. If an attL flanked cDNA library is generated from a cell or tissue of interest, that library can be recombined into the replicon plasmid in specifically in the sense orientation. A replicon expression library may be useful for expression clone screening that looks for genes introducing a new phenotype into a cell line, such as producing resistance to viral infection in a previously susceptible cell line. BP and LR recombination reactions appear to be scalable, so complex cDNA expression libraries can be generated with all of the inserts in the correct orientation for expression. The inclusion of a mutation at nsP2 amino acid 726 makes the replicon noncytopathic in mammalian cells ( and data not shown), which can allow a replicon cDNA expression library to be used in mammalian cells, although doing so appears to reduce the replication rate of the replicon. Coupling expression library replicons with an easily identified phenotypic readout, such as phenotype-induced GFP expression or altered cell viability, can be used to rapidly identify and enrich for cells with replicons expressing proteins affecting the phenotype. Once the cells of interest are enriched or isolated, the antisense-expression cassettes can be isolated from replicon RNA by reverse transcription, recombined into new entry plasmids, and either sequenced or recombined into new replicon plasmids for further rounds of screening. Because the replicons can incorporate and maintain fairly large inserts in plasmid form, it is possible that full-length genes can be directly isolated from antisense screening and immediately used for further testing. The percentage of cells that are infectable using our co-culture protocol is relatively high, increasing their utility in screening protocols.
Another interesting possibility is to use an antisense-expressing replicon library to screen for genes involved in particular cellular processes such as metabolism or viral infectability. Because replicons persistently replicate in cells, the expression of antisense RNA in selected replicon cells in theory could result in a "knock-out" phenotype. We have previously shown that expression of antisense RNA from a portion of the dengue virus genome drastically reduces the ability of cells and mosquitoes to be infected by dengue virus  and SINV mediated expression of antisense RNA to the Broad-Complex transcription factor in silkmoths was used to demonstrate the critical role of the protein in insect morphogenesis , and to assess the role of prophenoloxidase in mosquito melanization [22, 23]. SINV antisense expression is therefore a valuable tool for the functional evaluation of gene function in insects. Further evaluation of the utility of antisense expressing SINV replicons needs to be performed to determine their utility in reducing gene expression in insect cells.
The recombination-ready replicon expression plasmids detailed in this report provide convenient and efficient tools to the use of alphavirus replicons to address many biological questions in insect cells.
hepatitis delta virus
Green fluorescence protein
cytomegalovirus immediate early promoter
We thank the members of the AIDL for helpful discussions. This work was funded by NIH NIAID Grant AI046435-04 to K.O.
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