Introduction:
Stratagene is a well-known company, traditionally supplying reagents for molecular biology applications. In recent years Stratagene has expanded into the viral gene transfer market, offering systems based on AAV, Adenovirus and Retrovirus. The AdEasy system, developed by Bert Vogelstein’s group, is one of the most straightforward means to produce adenoviral vectors, with the recombination step taking place in bacteria and yielding a high percentage of positive viral clones. The helper-free AAV system offered by Stratagene also simplifies production of AAV vectors, a procedure that was quite cumbersome prior to the emergence of this technique. The ViraPort® Retroviral Gene Expression System works much the same way as the other retroviral systems offered by other companies described here, except that viral production is accomplished after a triple transient transfection in producer cells.
Technologies:
1. AdEasy
Adenoviruses are capable of infecting a broad range of cell types and infection is not dependent on active host cell division. Protein production techniques in mammalian cells require high efficiency gene delivery and high-level gene expression, both of which can be achieved using adenoviral vectors. Since adenoviral vectors have high expression levels, this system can be used for overexpressing recombinant proteins in mammalian cells. With expression in mammalian cells, the resulting proteins have the relevant posttranslational modifications and folding, which is not possible when overexpressing in prokaryotic systems.
The AdEasy system saves you a month of work over traditional methods by producing the recombinant adenoviral plasmid by homologous recombination in E.coli. Traditionally, two difficult and time consuming methods have been used to generate recombinant adenoviruses. The first method involves direct ligation of the gene of interest into the adenoviral genome. The scarcity of unique restriction sites and the prohibitive size of the genome (36 kb) make this method technically challenging. The second method, involves cloning the gene of interest into a shuttle vector and transferring the gene into the adenovirus genome by means of homologous recombination in mammalian cells. This method requires weeks of work in tissue culture performing multiple rounds of plaque isolations and is extremely laborious and time consuming.
In the AdEasy system, the gene of interest or expression cassette is first cloned into a shuttle vector. The resultant plasmid is contransformed into E. coli strain BJ5183 with the adenoviral backbone plasmid pAdEasy-1. Recombinant adenoviral plasmids are selected on kanamycin and confirmed by restriction digest. Lastly, the recombinant adenoviral plasmid is transfected into AD-293 cells where it is packaged into virus particles (figure below).
pAdEasy-1 Adenoviral Backbone
The pAdEasy-1 vector is a 33.4 kb plasmid containing most of the human adenovirus serotype 5 (Ad5) genome, with deletions in the genes E1 and E3. This vector has been developed to infect but not replicate in non-permissive target cells.
pShuttle Cloning Vectors
The pShuttle-CMV vector contains a multiple cloning site between the CMV promoter and the SV40 polyadenylation signal. The pShuttle vector contains only a multiple cloning site in which an entire expression cassette can be inserted. The regions indicated as arms are the stretches of sequence homology within the pAdEasy-1 vector, where homologous recombination occurs. Upon homologous recombination in bacteria with a plasmid containing the E1-E3 deleted Ad5 genome, a new plasmid is generated in which the expression cassette is inserted into the original E1 region of the adenovirus genome. This resulting plasmid is then transfected into HEK293 cells to generate a recombinant adenovirus expressing the gene product. Both these shuttle vectors contain the kanamycin resistance gene for the selection of recombinants. The RITR and LITR regions are short inverted repeats (right and left) which have a role in replication of the viral DNA.
Additional pShuttle Cloning Vectors with Vitality® hrGFP Reporter
Two shuttle vectors, pShuttle-IRES-hrGFP-1 and pShuttle-IRES-hrGFP-2 are available separately. Both vectors contain the CMV promoter and a bicistronic expression cassette in which the multiple cloning site (MCS) is followed by the EMCV-IRES, which directs translation of the Vitality humanized Renilla GFP (hrGFP). This design allows the expression of the gene of interest to be monitored at the single-cell level due to expression of the hrGFP on the same transcript. The gene of interest may be fused to three contiguous copies of either the FLAG® epitope or the hemagglutanin (HA) epitope.
pShuttle-CMV-lacZ Control Vector
The lacZ gene was inserted in the multiple cloning site of the pShuttle-CMV to produce pShuttle-CMV-lacZ. This construct is provided as a control for the production of recombinant adenovirus.
2. AdEasy-XL
Like the original system, the AdEasy XL system saves you a month of work over traditional methods by producing the recombinant adenoviral plasmid by homologous recombination in E.coli. The difference is that AdEasy XL system uses specialty competent cells, BJ5183-AD-1 cells, which are pre-transformed with the adenoviral backbone (pAdEasy-1 plasmid). This reduces the cotransformation of both the shuttle vector and large adenoviral backbone in the original system to a single transformation of the shuttle vector containing your gene of interest.
The gene of interest or expression cassette is first cloned into a shuttle vector. The resultant plasmid is transformed into the specialty E. coli strain BJ5183-AD-1 containing the adenoviral backbone plasmid pAdEasy-1. Recombinant adenoviral plasmids are selected on kanamycin and confirmed by restriction digest. Lastly, the recombinant adenoviral plasmid is transfected into AD-293 cells where it is packaged into virus particles.
3. AAV Helper-Free System
Expressing genes of interest with the AAV Helper-Free system consists of a three-plasmid cotransfection of a packaging cell line, collection of high-titer recombinant virus from the cell lysates, and infection of target cells for expression studies. As shown in the flowchart below, the pAAV-LacZ vector with the insert-containing expression cassette is cotransfected into a packaging cell line along with the 7.3-kb pAAV-RC vector and the 11.6-kb pHelper vector. The pAAV-RC vector encodes the rep and cap DNA replication proteins required to make infectious virions, and the pHelper vector encodes the adenovirus genes required for producing high-titer AAV-2 in HEK293 cells. The HEK293 cell line contributes adenovirus E1A and E1B proteins, which are stably expressed and essential for packaging virions. From this three-plasmid cotransfection, recombinant, replication-deficient AAV-2 virions are produced at titers that approach and are often in excess of 107 vp/ml. If higher titers are desired, stocks can be concentrated; titers as high as >1011 - 1012 vp/ml have been published.
High-titer recombinant AAV-2 stocks are used to infect target cells. The AAV genome typically remains epichromosomal, often forming high molecular-weight concatemers. In cell populations that are dividing slowly or are static, long-term expression will be achieved from cells where the virus exists epichromosomally. In rapidly dviding cells, the epichromosmal genome will be lost over time. However, integration of the AAV genome into the host cell chromosome can occur, and you can maintain long-term gene expression in dividing cell populations. Integration events, however, are rare. By using extremely high multiplicity of infection (MOI) or by supplying adenoviral replicase, the frequency of viral integration can be increased. Note that using wild-type adenovirus to increase integration frequency lowers to biosafetly of the AAV system.
pCMV-MCS Vector
The pCMV-MCS is a 4.5 kb cloning vector. The gene of interest is cloned into the multiple cloning site. After cloning, the expression cassette is removed from this vector using the flanking NotI sites for subsequent cloning into the pAAV-LacZ vector.
pAAV-LacZ Vector
The 7.3 kb pAAV-LacZ is the replication-deficient AAV vector. This vector serves two purposes; first, as a cloning vector; second, as a control vector. As a cloning vector, this vector is cut with NotI, the LacZ cassette is lost and the expression cassette, harboring the gene of interest, from the pCMV-MCS vector will be cloned into the complementing NotI sites of this vector. The recombinant vector will be cotransfected into a viral packaging cell line for production of recombinant, replication-deficient AAV virions. This vector is also a control using the LacZ reporter enzyme.
pAAV-RC Vector
This 7.3 kb vector harbors the cap and rep genes that make the capsid and DNA replication proteins required to make infectious virions. This vector is cotransfected into the packaging cell line for production of recombinant, replication-deficient AAV virions.
pHelper Vector
This 11.6 kb vector carries the adenovirus genes (E2A, E4 and VA RNAs) required for inducing the lytic phase of AAV. This vector is cotransfected into the packaging cell line to produce recombinant, replication-deficient AAV virions.
ViraPack™ Transfection Kit
The ViraPack™ Transfection Kit is available separately from the AAV helper-free system. The kit is developed specifically for the transfection of viral plasmids into HEK293 cells.
AAV-293 Cells
The AAV-293 cells are a strain of HEK-293 cells that have been optimized specifically for the packaging of AAV virions.
AAV-HT1080 Cells
The AAV-HT1080 cell line is an important component in the titering step of the virus.
pAAV-IRES-hrGFP Vector
The vector pAAV-IRES-hrGFP contains a bicistronic expression cassette in which the Vitality® hrGFP gene is expressed as a second open reading frame downstream of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The hrGFP gene serves as a useful marker for monitoring the transfection efficiency of the HEK 293 cells for viral production. Another unique feature of this vector is the FLAG sequence (3x) at the c-terminus of MCS. The foreign gene could be cloned in frame with the tag and the protein could be further purified or detected by anti-FLAG antibody.
pAAV-hrGFP Vector
The pAAV-hrGFP is a nice alternative to the pAAV-LacZ positive control vector. This vector allows you to confirm the transfection efficiency of your packaging cell line in vivo using the Vitality hrGFP reporter gene. This reporter gene allows for easy detection of expression in vivo using fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis.
4. ViraPort® Retroviral Gene Expression System
The ViraPort® retroviral gene expression system* is a comprehensive line of gene delivery products providing transduction efficiencies approaching 100% in a wide range of mitotic cells. With this system, gene copy number per cell can be controlled by adjusting the multiplicity of infection, enabling you to clone a single gene by its function. Available with the system are high-titer cloning vectors, specialized vectors that provide the necessary components to make infectious virions, and premade libraries that allow you to clone a single gene by its function.
The pFB and pFBNeo retroviral expression vectors, based on a modified MMLV, are the foundation of the ViraPort system. Packaging sequences have been extended, resulting in extremely high titers. Genes coding for gag/pol and env have been transferred from the viral genome to the pVPack-GP vector+ and one of four pVPack env-expressing vectors+, which share no regions of homology with the pFB vectors. This eliminates the generation of replication-competent, wild-type virus.
Stratagene's pVPack vector system comprises a set of 5 vectors that can be used with any MMLV-based retroviral vector to produce viral supernatants that have titers that are consistently > 107 colony forming units (cfu)/ml in transient triple-transfection experiments. The vectors include a gag-pol-expressing vector (pVPack-GP vector) that is cotransfected with the retroviral expression vector together with one of a choice of 4 envelope (env)-expressing vectors. The choice of the env-expressing vector is based on the range of cell types the user wishes to transduce.
With the pVPack vector system, all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, with minimal or no sequence overlap between the plasmids. This makes the pVPack system much safer than the majority of stable producer cell lines or vector-based systems for which there is a large degree of homology between the packaging vectors and the retroviral expression vector. In other systems there is a relatively high probability of production of replication-competent retrovirus (RCR) due to homologous recombination between the vectors.
The pVPack vector system offers 4 different env-expressing vectors:
1. The pVPack-Eco vector is the safest vector because it will only efficiently transduce mouse or rat cells. The ecotropic virus infects human cells with extremely low efficiencies.
2. The amphotropic envelope protein has historically been the protein of choice for infection of human and other mammalian cell lines.
3. More recently, the 10A1 envelope protein has been used due to its increased versatility relative to the amphotropic protein. The 10A1 protein can infect any cell that an amphotropic virus can infect, but because it recognizes a second receptor, the transduction efficiency can be higher than efficiencies with the amphotropic envelope proteins. The ecotropic, amphotropic and 10A1 proteins are all natural MMLV variants, and are all relatively labile and thus considered relatively safe compared with other viral systems.
4. The vesicular stomatitis virus G protein (VSV-G) is rapidly becoming the most popular envelope protein. Unlike the other three MMLV-derived envelope proteins which recognize cell surface receptors, VSV-G recognizes a phospholipid that is present on all cell types, and thus can theoretically allow the efficient infection of any mitotic cell. Special precautions must be used when working with this vector. VSV-G has the added advantage that it confers a stability to the retrovirus that allows viral supernatants to be concentrated to 2000-fold or more by ultracentrifugation; this cannot be done with the other three more labile viruses.
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