Lentivirus FAQs

How should I store the viral particles (lentivirus, AAV or VSV) that I just received?

Our viral products are shipped with dry ice. If there is no dry ice left in your shipment upon arrival, please let us know at once. For long term storage, it is recommended to store the viral particles at -80°C for up to 12 months from date of receipt. Avoid repeated freeze-thaw cycles as titers can drop significantly with each freeze-thaw cycle.

I ordered off-the-shelf viral particles and kept them at -80°C as instructed. How do I thaw the virus?

Thaw at room temperature and add directly to the cells. If an intermediate dilution is needed, the virus can be diluted in the cell culture medium at room temperature.
Titers can drop significantly with each freeze-thaw cycle. If the virus preparation is going to be used multiple times, it is best to prepare very small aliquots and freeze them immediately. Avoid multiple freeze/thaw cycles.

What are the advantages of lentivirus transduction over lipid-mediated transfection?

Lentivirus transduction is a powerful tool for introducing genes into cells and has a few advantages over lipid-mediated transfection, particularly in terms of efficiency, stability, cell type versatility, and cargo capacity.

Efficiency: Lentivirus transduction is more efficient than lipid-mediated transfection, meaning that it can introduce genetic material into a higher percentage of cells. This is because lentiviruses have evolved to efficiently enter and integrate into the host cell's genome. Thus, they are especially useful to transduce cells that are somewhat resistant to lipid-mediated transfection.
Stable integration: Lentiviruses can integrate into the genome, resulting in stable and long-lasting expression of the introduced gene. Lipid-mediated transfection typically results in transient expression and integration is a rare event (even when using linearized DNA).
Versatility: Lentivirus transduction is used to introduce genes into a wide range of cell types, including both dividing and non-dividing cells. In contrast, lipid-mediated transfection is less efficient in primary cells or non-dividing cells.
Lower toxicity: Lentivirus transduction is less toxic to cells than lipid-mediated transfection.
Larger cargo capacity: Lentiviruses can carry larger genetic payloads than lipid-mediated transfection methods, allowing for the introduction of more complex genetic constructs.

What is a pseudovirus?

The most commonly used lentivirus is based on the HIV virus that infects human lymphocytes through the specific interaction of its envelope protein gp120 with the CD4 receptor. In biotechnology, gp120 is replaced by another envelope glycoprotein, typically VSV-G (Vesicular stomatitis virus G protein), which binds to the human LDL receptor (low-density lipoprotein receptor) expressed on most cell types. An HIV-based lentivirus containing an envelope protein other than gp120 is called a pseudotyped virus, or pseudovirus. Pseudoviruses in which the envelope protein has been replaced by the SARS-CoV-2 Spike protein will bind to human ACE2 and are useful to study Spike/ACE2-mediated infection.
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What are bald lentiviruses?

Bald lentiviruses are pseudovirus particles in which the envelope protein has been removed entirely. Therefore, the bald pseudovirus cannot interact with host cells in a receptor-specific fashion. Bald lentiviruses serve as a negative control (or blank) in infection experiments.
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What are reporter lentiviruses?

Reporter lentiviruses contain a plasmid to express a reporter gene such as GFP or Luciferase in the target cell. This reporter gene can be under the control of a constitutive promoter (e.g. CMV) or under the control of a promoter containing a specific response element to study the activation of a particular transcription factor or signaling pathway. The reporter gene is transduced when the lentivirus enters the target cell and the resulting expression of the reporter protein can be detected by fluorescence (GFP) or luminescence (luciferase activity). SARS-CoV-2 Spike reporter pseudoviruses can be used to quantify Spike/ACE2-mediated infection.
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What safety level do you need to use BPS lentiviruses?

A biosafety level 2 facility is sufficient to use BPS lentiviruses. Our replication-incompetent lentivirus particles, produced from packaging cells, contain minimal genetic information that includes the gene of interest but does not include viral genes involved in packaging. Therefore the virus packaging genes are not expressed in the transduced cells and cells infected with our viruses cannot produce new viral particles.

What types of cells can you infect with a lentivirus?

Lentiviruses infect most cell types and can transduce dividing cells as well as quiescent cells, whether they are immortalized or primary cells, which makes them particularly suitable to use with cells that are difficult to transfect using other methods.

What is a non-integrating lentivirus?

A lentiviral genome integrates in the host DNA using an enzyme called retroviral integrase. This enzyme has been deactivated in BPS Bioscience’s integration-deficient lentiviruses, therefore the DNA carried by the virus cannot integrate into the host DNA. The lack of integration prevents possible off-target effects resulting from the random genetic disruption in CRISPR/Cas9 knock-out cells. They are used for transient transduction or as control for integrating viruses.

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What is an MOI?

Multiplicity Of Infection (MOI) is the ratio of the number of infective virions to use per number of target cells. For example, if 100,000 infective particles are added to 1 million cells, the MOI is 0.1. The optimal MOI needs to be determined for each cell line. Optimization is easily carried out using a GFP reporter lentivirus (BPS Bioscience, #79979). Suggested MOIs for commonly used cells are indicated in the following table:

Cell Line	HEK293	HeLa	HCT116	Jurkat	MCF7	MDA-MB-231	A549
Suggested MOI	5	3	5	10	2	1	5

What are good cellular models to use BPS Bioscience Spike (SARS-CoV-2) pseudoviruses?

Spike interacts with human cells by binding to membrane receptor ACE2 (angiotensin converting enzyme 2), therefore cells expressing high levels of ACE2 are good models. For best results we recommend using ACE2-overexpressing cells such as ACE2-HeLa, ACE2-CHO and ACE2-HEK293 cells, in which ACE2 overexpression has been validated by Flow Cytometry. Vero E6 cells are also commonly used for coronavirus studies and do express ACE2 endogenously. However, based on experiments performed by BPS scientists, ACE2-HEK293 cells provide greater transduction when compared to Vero E6 cells. To optimize Vero E6-based experiments, BPS recommends using TMPRSS2-Vero E6 cells. These cells express serine protease TMPRRS2 that cleaves the S2 subunit of Spike and promotes membrane fusion and viral entry into the host cell.
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What is the best AAV serotype for my cell type?

So far, 11 AAV serotypes have been isolated from humans and non-human primates. Each serotype shows preferential binding for particular cell types and tissues, as determined by the binding affinity of the capsid proteins to receptors on the cell surface (see tables below). Scientists can use this tropism to efficiently target specific cell types.
Genetically engineered AAV serotypes have been developed to further increase tissue tropism and transduction efficiency. For example, AAV-DJ is a synthetic serotype made from eight different wild-type AAV serotypes (AAV2, 4, 5, 8, 9, avian, bovine, and goat AAV) using DNA shuffling. The AAV-DJ serotype can infect a broad range of cell types and has better transduction efficiency in vitro and in vivo compared to wild-type serotypes.
A survey of in vitro transduction efficiency for serotypes 1 to 9 has been published (Ellis BL, et al. 2013)

Serotype	Tissue
AAV1	CNS, Heart, and Skeletal Muscle
AAV2	CNS and Kidney
AAV3	Liver
AAV4	CNS and Lung
AAV5	CNS and Lung
AAV6	Lung and Skeletal Muscle
AAV7	Liver and Skeletal Muscle
AAV8	CNS, Heart, Liver, Pancreas, and Skeletal Muscle
AAV9	CNS, Heart, Liver, Lung, and Skeletal Muscle

What safety level do you need to use AAVs?

AAVs require the use of a Biosafety Level 1 facility. BPS Bioscience recommends following all local, federal, state, and institutional regulations and using all appropriate safety precautions.
AAVs are replication-defective, non-pathogenic, and have not been associated with any human disease. Wild-type AAVs integrate into the host genome at negligible frequencies. When it occurs, integration is predominantly at the “safe harbor” site AAVS1 on the human chromosome 19. However, recombinant AAV vectors exist as episomes inside the cells and therefore do not disrupt the genome.

What is the titer of the supplied AAVs?

Our AAVs are supplied as two vials (50 µl x 2) of AAV at a titer ≥1 x 10¹² vector genomes (vg)/ml. Therefore, each vial contains approximately 50 billion vector genomes. The titer is determined by qPCR and varies with each lot; the exact value is provided with each shipment.

What kind of quality control do you perform on AAVs?

Each AAV preparation is purified by iodixanol density gradient and ultra-centrifugation. The titer is determined by SYBR Green qPCR. Titers vary with each lot and the exact titer value is provided with each shipment. The purity of the AAV particles is determined for each lot by staining on a 4-20% SDS-PAGE gel using One-Step Lumitein™ UV Protein Gel Stain (Biotium #21005) and is guaranteed to be greater than 90%. If the AAV contains a fluorescent reporter gene, it is functionally confirmed by transducing HEK293 cells and detecting the resulting fluorescence using a fluorescence microscope. For luciferase reporter AAVs, HEK293 cells are transduced, and the luciferase activity is measured using the ONE-Step™ Luciferase Assay System (BPS Bioscience #60690). Finally, SaCas9 AAVs are tested by transducing HEK293 cells and confirming HA-tagged SaCas9 expression by Western blot using an anti-HA-tag antibody.

How do I thaw my AAVs?

Keep AAV frozen -80°C until use. Thaw the AAV particles on ice. Quickly centrifuge the tube to recover its full content. Gentle vortex mixing of the tube is also acceptable. The viral particles may be diluted in cell culture medium if desired or can be added directly to the cells. Alternatively, AAVs can be diluted in Phosphate Buffered Saline (PBS) for in vivo use.
AAVs are sensitive to freeze/thaw cycles. The viral titer will drop sharply with each freeze/thaw cycle.

Can I use AAVs with confluent cells?

Most adherent cells are more efficiently transduced when they reach approximately 70% confluency. However, AAVs can also be used with cells at >70% confluency provided that the experimental conditions have been optimized accordingly (for example, by increasing the Multiplicity of Infection).

Can I use AAVs with Primary cells?

Yes. In fact, AAV transduction works better than other methods of transfection or viral transduction when using primary and non-dividing cells, including induced pluripotent stem (iPS) cells and iPS-derived cells.

Can I generate a stable cell line using AAV?

No. AAVs do not integrate into the genome and are not suitable to generate stable cell lines. Transduced genes are maintained in the cells as episomes and can be expressed for up to 6 months or longer in non-dividing cells, or up to 3 months in dividing cells (depending on the cell type and MOI used). However, expression is diluted over time in dividing cells.

What is T2A?

The T2A linker is a self-cleaving peptide derived from Thosea asigna virus 2A that is used to express two reporter proteins at equal levels of expression. For example, our dual-reporter AAV systems such as AAV luciferase-eGFP use the DNA construct “Luciferase-T2A-eGFP” under the control of a single promoter. The product of this DNA construct is a unique transcript which encodes the protein “Luciferase-T2A-GFP”. Linker T2A undergoes self-cleavage, leading to the release of luciferase and eGFP as two separate proteins in roughly equal proportions.

Can I use AAVs from BPS Bioscience for in vivo experimentation?

Yes, BPS Bioscience’s AAVs can be used in animal research and may be injected into laboratory animals. Our purification process removes empty capsids from AAV preparations and guaranties high quality AAV particles. If necessary, users may request further purification or testing as a custom order.
Please note that our products are not for use in humans or for diagnostic or therapeutic applications.

How do I obtain the best possible efficacy of transduction?

Reporter AAVs can be used to optimize transduction efficiencies in your cell line of interest. Particle-to-cell ratio is the most critical parameter to vary when optimizing protein expression in the target cells. Adjusting the incubation volume is the next best parameter to optimize. Adjusting the temperature or the cell density (if adherent cells are being transduced) may also improve transduction efficiencies. For adherent cells, we recommend a confluency of about 70%.

What are the SaCas9 AAVs?

SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high nuclease efficiency in mammalian cells, and its small size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5’-NNGRRT-3’, than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. They are used to constitutively express SaCas9 in the target cells to genetically engineer the cells using CRISPR/Cas9 technology.

What information is needed when ordering a custom AAV design or custom AAV packaging service?

Our team of scientific experts is available to help with the custom design or your AAVs. To expedite the process, these are the points to consider.

For custom AAV design:

If you have a gene template, please provide your gene template to us.
If you would like us to synthesize a gene without a template, please provide the gene number, sequence map, species, and gene region.
To generate AAVs for the manipulation of gene expression using techniques such as RNAi or CRISPR, please provide us with target gene information such as database accession number, species, and gene length (ideally, under 3.2 kb).
Please indicate if you prefer a specific promoter to be used in the construct.
Please indicate if you would prefer to use a particular reporter gene.
If two or more genes will be co-expressed, please provide all relevant information for each gene.

For custom packaging services:

Please indicate which AAV serotype you will use. Our team is available to guide your selection if needed.
If it is unknow which serotype is capable of infecting your cells, we offer reporter AAVs for the comparison of various serotypes to help you determine the optimal serotype for your experiments.
If providing your own plasmid for packaging, confirm ITR spacing and fully sequence the plasmid to avoid possible complications associated with common mutations driven by the presence of the ITRs. Please send us the vector map and the full sequence of the plasmid.
When placing your custom order, please indicate the desired amount, titer, and packaging requirements for your deliverable AAV.

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