Choose Lentivirus as your viral delivery system if:

• Your expression cassette is smaller than 8 kb.
• You want your gene integrated into the host genome for long term, stable expression.
• You need a vector that has low immunogenicity.
• You wish to infect a large proportion of cells without requiring a high titer of virus.
• You wish to perform a genome-wide functional study, in vivo imaging study, or produce iPSCs.
  

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Lentiviruses are a particular group of retroviruses that are able to infect both dividing and non-dividing cells. Recombinant lentiviruses have been specially altered to be capable of infecting a broad host range by a process called pseudotyping.

The main advantage of using lentivirus in your experiment is its ability to integrate a part of its genome into the host cell’s DNA. This ensures long term expression in a cell population, as the viral DNA will be replicated with host DNA when the cell divides. Additionally, lentivirus tends to integrate in favourable locations for transcription, leading to high levels of transgene expression. Lentivirus’ ability to integrate makes it ideal for in vivo imaging studies and creating induced pluripotent stem cells (iPSCs). This ability, however, may not be desired for certain applications. Insertional mutagenesis has the potential to disrupt normal gene function and promote oncogenesis.

Lentivirus is able to package 8 kb of genetic material, has low immunogenicity, and high infection efficiency. Lentivirus is one of the most commonly used expression systems, so there are a huge variety of ready-made vectors available for use. As well, they are very commonly used in genome-wide functional studies, so there are a wide range of pooled lentiviral libraries commercially available, including cDNA, shRNA, and gRNA libraries.

Choose Retrovirus as your viral delivery system if:

• You wish to infect only dividing cells.
• Your expression cassette is smaller than 8 kb.
• You want your gene integrated into the host genome for long term, stable expression in dividing cells.

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When scientists refer to 'retrovirus' as a gene delivery method, they are usually referring to the subset of retroviruses called gamma-retroviruses. Gamma-retrovirus are distinguished from lentiviruses (another genus within Retroviridae) in that they can only infect dividing cells, while lentivirus can infect both dividing and non-dividing cells. For more information on lentiviruses, click here.

Retrovirus is a ssRNA virus with the ability to package 8 kb of genetic material that is integrated into the DNA of dividing cells. This means retroviruses can be used to ensure long term expression in a cell population, since the viral DNA will be replicated along with host DNA when the cell divides. However, retroviruses cannot be used to infect cell types that do not divide frequently, such as neurons or hematopoietic stem cells.

One potential problem with using retroviruses in gene therapy studies is that integration is random and can occur at any point in the host genome. Depending on where integration occurs, it may lead to insertional mutagenesis and oncogenesis.

Choose Baculovirus as your expression system if:

• Your gene insert is larger than 8 kb, or you wish to express a multiprotein complex.
• You wish your proteins to have post-translational modifications such as phosphorylation, glycosylation, and acylation.
• You wish to produce large quantities of recombinant protein.
• You wish to work in BSL-1 (Biosafety level 1) facilities.

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Baculovirus is a large virus with a circular, double stranded genome of 80 to 180 kb. It naturally infects insects such as butterfly and moth larvae. Baculovirus has a broad host range, but must be cultivated in insect cells for packaging to occur. Once cultivated, it can be easily produced and purified at high titres.

A few attributes make this a very appealing prospect for gene expression, particularly for the production of recombinant protein. First, the gene capacity of baculovirus is, in theory, unlimited, as the capsid can freely extend. This makes it ideal for expressing large transgenes or multiprotein complexes. Secondly, insect cells can perform post-translational modifications that bacteria cannot, while being easier to grow than mammalian cells. Finally, it is easy to scale up recombinant protein production in order to generate large amounts of protein quickly and efficiently.

Proteins produced using the baculovirus system include those used in the FDA- and EMA-approved vaccines Cervarix®, Provenge® and FluBlok®. Baculovirus has been used in a variety of other applications, including gene therapy, drug screening, eukaryotic surface display, and even the production of other gene transfer vectors such as AAVs and lentiviruses.

Choose AAV as your viral delivery system if:

• Your expression cassette is smaller than 4.7 kb.
• You are performing an in vivo experiment.
• You do not want your gene integrated into the host genome.
• You need a vector that has low immunogenicity.
• You wish to infect only a few, specific cell types.

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The Adeno-Associated Virus (AAV) is a promising candidate for virus-based gene therapy. Recombinant AAV forms episomal circular concatemers in the nucleus of cells. This episome does not integrate into the host genome, so as the cells replicate, the DNA becomes diluted. This will eventually result in the loss of transgene expression, with the rate of loss dependent on the replication rate of the transduced cell. However, the fact that AAV doesn’t integrate means that there is no risk of insertional mutagenesis, which can lead to oncogenesis. Additionally, of the commonly used viral vectors, AAV elicits the lowest immune response. For these reasons, AAV is often used for therapeutic applications and in vivo experiments.

Many different serotypes of AAV have been discovered. These serotypes have different cell surface receptors, which leads to a different tropism for each virus variant. This natural variation can be exploited in gene therapy; one can choose to package a recombinant AAV as a serotype that preferentially infects the cell type(s) of interest. Click here for more information on the tropism of different AAV serotypes.

A drawback to using AAV is its small packaging size. The length of the expression cassette should not exceed 4.7 kb. Anything above this size leads to a considerable reduction in viral yields and/or truncations of the transgene. Another disadvantage of AAV is that some cell types may require a high Multiplicity of Infection (MOI) to achieve transduction.

Choose HSV as your viral delivery system if

• Your gene insert is larger than 8 kb.
• You do not want your gene integrated into the host genome.
• You wish to infect neuronal cells for long term expression without integration.
• You require a highly infectious virus.

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Herpes Simplex Virus (HSV) is a neurotrophic virus with a large (152 kb) dsDNA genome. HSV does not integrate into the host DNA, but is known to establish long-term latent infections by remaining as a chromatinzed episome in the host nucleus. HSV has a broad host cell range, including non-dividing cells that may normally be difficult to transduce, such as neuronal cells.

Due to its large genome size, HSV is an excellent choice for delivering transgenes that would be too large to package in other viruses. It is particularly useful for targeting neuronal cells as it is able to establish stable long term gene expression in neurons, without integration. It also has the interesting ability to be transported within neurons and across synapses. This property can be exploited to trace neuronal pathways by introducing the vector into peripheral sites like skin or muscle then locating transgene expression in the neuronal cell bodies which connect to those sites.

Choose Adenovirus as your viral delivery system if:

• Your insert cassette is smaller than 8 kb.
• You do not want your gene integrated into the host genome.
• You need high levels of transient expression.
• You require a high transduction efficiency.
• You wish to infect with a high titer of virus.

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Adenoviruses are medium-sized viruses with a linear dsDNA genome that is ~36 kb in length. Of this 36 kb, 8 kb can be used for carrying an expression cassette. Adenoviral DNA does not integrate into its host cell’s DNA. The DNA exists in the nucleus of the host cell, but is not replicated when the cell undergoes cell division. As the cells replicate over time, the adenoviral DNA expression weakens and eventually will disappear. Therefore, adenovirus is not a good choice for experiments that require long term, stable expression.

However, due to its excellent transduction efficiency (almost 100%) and high level of gene expression, adenovirus has been a popular choice for gene and shRNA expression for a variety of applications. These include vaccine production, gene therapy, gene knockdown, and the production of membrane proteins and antibodies. Adenoviruses have been already used in several gene therapy trials, particularly those targeting cancer cells. The first gene therapy product ever licensed was an adenovirus carrying the p53 repressor gene, for the treatment of head and neck squamous cell carcinoma.

While infection with adenovirus offers several advantages, the cloning of these viruses can be difficult and time consuming. Traditional methods involve the cloning of the transgene into a shuttle vector, then transferring the transgene into the adenoviral vector. As well, the adenoviral vector must be linearized before it can be packaged.

Choose a Protein Vector for bacterial expression if:

• You need to purify large quantities of protein
• You do not require post-translational modifications to the protein
• You do not have the equipment to culture mammalian cells
• You do not wish to use a viral system

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Protein vectors are a non-viral option for expressing protein to be purified. Protein vectors include a purification tag that will be fused to the gene of interest, making it easy to purify the protein once it is expressed. To prevent potential interference of the purification tag with normal protein function, some tags are able to be cleaved from the protein of interest using proteolytic agents such as thrombin or TEV protease.

Purification tags may be fused to either the C- or N-terminal of a protein, and can work in a variety of ways. Affinity tags allow for proteins to be purified using an affinity technique. Examples include maltose binding protein (MBP), glutathione-S-transferase (GST), and His tags. Epitope tags can be used to purify proteins using high-affinity antibodies which bind to them. Examples include HA tags, and FLAG tags (D tags). As well, certain tags may help the protein of interest to fold correctly and keep it from precipitating. These are called solubilization tags, and examples include MBP and GST.

When a mammalian gene is translated in mammalian cells, it will undergo all post-translational modifications that would occur in nature. Therefore protein produced in a mammalian system will be more authentic for experimental use than one produced in a bacterial system. However, mammalian cells are much more difficult and time-consuming to culture than bacterial cells. Therefore, for applications where large amounts of protein are required and authenticity is not a major concern, bacterial expression is a good choice. If large amounts of post-translationally processed protein are required, a baculoviral expression system should be considered.

For a transgene to be expressed in bacteria, it must be driven by a bacterial promoter. Common bacterial promoters that are used in protein vectors include T7 and Lac.

Choose a Protein Vector for mammalian expression if:

• You wish for your protein to have post-translational modifications
• You have the equipment to culture mammalian cells
• You do not want to use a viral system

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Protein vectors are a non-viral option for expressing protein to be purified. Protein vectors include a purification tag that will be fused to the gene of interest, making it easy to purify the protein once it is expressed. To prevent potential interference of the purification tag with normal protein function, some tags are able to be cleaved from the protein of interest using proteolytic agents such as thrombin or TEV protease.

Purification tags may be fused to either the C- or N-terminal of a protein, and can work in a variety of ways. Affinity tags allow for proteins to be purified using an affinity technique. Examples include maltose binding protein (MBP), glutathione-S-transferase (GST), and His tags. Epitope tags can be used to purify proteins using high-affinity antibodies which bind to them. Examples include HA tags, and FLAG tags (D tags). As well, certain tags may help the protein of interest to fold correctly and keep it from precipitating. These are called solubilization tags, and include MBP and GST.

When a mammalian gene is translated in mammalian cells, it will undergo all post-translational modifications that would occur in nature. Therefore protein produced in a mammalian system will be more authentic for experimental use than one produced in a bacterial system. However, mammalian cells are much more difficult and time-consuming to culture than bacterial cells. So, for applications where post-translational processing is important, mammalian expression is a good choice. If large amounts of post-translationally processed protein are required, a baculoviral expression system should be considered.

For a transgene to be expressed in mammalian cells, it must be driven by a mammalian promoter. Common mammalian promoters that are used in protein vectors include CMV, EF1a, CAG, and EF1a.

Choose an ORF Vector if:

• You wish to clone a gene into your own custom vector
• You require a gene as template for PCR
• You wish to work with the same gene in many different vectors

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For doing your own cloning, it’s best to work from a simple non-viral plasmid carrying your gene of interest. These are called Open Reading Frame (ORF) vectors, as they contain only the translated region of the gene without the 5’ or 3’ untranslated regions. They also lack extraneous elements such as promoters or reporters.

An ideal ORF vector is one that has a variety of restriction enzyme sites available on either side of the insert. This allows it to be easily cloned into an assortment of new vectors using traditional ligation. However, even without convenient restriction sites, an ORF can be used for ligation-free cloning by amplifying it via PCR.

There are a few advantages of working from an ORF vector instead of a synthesized gene. First, an ORF vector can be easily amplified via bacterial transformation, so there is no risk of running out of DNA. Second, large genes may be less expensive to purchase as an ORF vector than they would be to synthesize. Finally, it can be difficult to synthesize sequences that contain repeated elements or have a high GC-content; for these sequences, it would be easier to use an ORF vector.

abm carries a wide range of viral vectors for use in CRISPR Cas9 experiments. For detailed recommendations on which products to use for these applications check out our CRISPR Experimental Design Tool.

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Choose siRNA Lentivirus if:

You want your siRNA integrated into the host genome for long term, stable expression.

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Our specially designed iLenti™ vectors offer several advantages:

• Uses more potent 27-29 bp oligos than the traditional 19 or 21 bp oligos.
• Convergent promoter design avoids hairpin loop structure, simplifying sequencing and amplification.
• GFP reporter available for monitoring transfection/infection.
• Buy 4 siRNAs at a discount! siRNA lentiviruses are available individually or in groups of 4.

Choose siRNA AAV if:

You do not want your siRNA integrated into the host genome, and you wish to work in a viral system.

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Our specially designed siRNA vectors offer several advantages:

• Uses more potent 27-29 bp oligos than the traditional 19 or 21 bp oligos.
• Convergent promoter design avoids hairpin loop structure, simplifying sequencing and amplification.
• GFP reporter for monitoring transfection/infection.
• Four vectors/viruses targeting your gene of interest are pooled for increased knockdown of gene expression.

Choose siRNA oligos if:

You do not want your siRNA integrated into the host genome, and you wish to work without any viral elements.

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Our siRNA oligos offer several advantages:

• Produced under an ISO9000 quality system.
• HPLC purified: siRNA content >97%.
• Chemically modified (2’-OMe) for increased stability, higher efficacy, and lower toxicity in vitro and in vivo.
• Set of 3 siRNA oligo duplexes for increased gene silencing.
• Free positive and negative controls included in every set.
• No extraneous viral elements.

Choose miRNA mimics or agomirs if:

You do not want your miRNA integrated into the host genome, and you wish to work without any viral elements.

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Our miRNA mimics and agomirs offer several advantages:

• Chemically modified dsRNA designed to copy the functionality of mature endogenous miRNA.
• Agomirs are chemically engineered to enhance transfection efficiency and resistance to RNAses.
• Mimics/agomirs are offered for mature miRNA and precursors.
• No extraneous viral elements.

Choose miRNA Lentivirus if:

You want your miRNA integrated into the host genome for long term, stable expression.

Choose miRNA AAV if:

You do not want your miRNA integrated into host DNA and you are performing an in vivo experiment.

Choose miRNA Adenovirus if:

You do not want your miRNA integrated into host DNA and you require high levels of expression.

Choose miRNA Inhibitors and Antagomirs if:

You do not want your miRNA inhibitor integrated into the host genome, and you wish to work without any viral elements.

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Our miRNA inhibitors and antagomirs offer several advantages:

• Chemically synthesized single stranded oligonucleotides designed to bind and inhibit miRNA.
• Antagomirs are chemically engineered to enhance transfection efficiency and prevent degradation.
• No extraneous viral elements.

Choose miRNA Inhibitor Lentivirus if:

You want your miRNA inhibitor integrated into the host genome for long term, stable expression.

Choose miRNA Inhibitor AAV if:

You do not want your miRNA inhibitor integrated into host DNA and you are performing an in vivo experiment.

Choose miRNA Inhibitor Adenovirus if:

You do not want your miRNA inhibitor integrated into host DNA and you require high levels of expression.