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Addgene

Biosafety Resource Guide

The NIH provides basic guidelines to ensure that research is being performed within the highest standards possible to protect researchers. Visit the NIH's website for more details about safety considerations. Specific questions about how biosafety relates to your research should be directed to your institution’s Biosafety Committee.

Documentation

Addgene has worked closely with safety consultants to ensure that we provide the relevant safety forms for each order. We do not provide Material Safety Data Sheets (MSDS), which are now called Safety Data Sheets (SDS) in the US, because plasmids shipped as bacterial stabs, plasmids shipped as purified DNA, and replication-defective viral particles are not classified as chemicals (only chemicals need an SDS, per the Hazard Communication Standard). If your organization still requires a SDS due to internal regulations, please download the appropriate SDS for your material. US Department of Transportation (DOT) and International Air Transport Association (IATA) forms are included as needed.

Viral Vector Biosafety Resources

The ability of viruses to target and invade cells makes them a key tool for biological research, but also raises biosafety concerns. Chief concerns are 1) the potential generation of replication competent virus and 2) the potential for oncogenesis through insertional mutagenesis. These risks are dependent on the vector system used and the transgene insert encoded by the vector. While the viral vector systems in current use are designed to minimize risk to researchers, it’s important to consider both of these concerns when designing your experiments.

The NIH provides basic guidelines for working with various types of vectors; however, these guidelines may differ depending on the insert type and envelope used ( see Vector Characteristics Impacting Biosafety). Please consult with your institution’s Biosafety Committee before beginning virus work.

General Biosafety Guidelines

  • Adenovirus: This virus can cause mild to severe respiratory disease in humans. Ethanol cleaning does not inactivate adenovirus; 10% bleach (0.5% sodium hypochlorite) should be used instead. Use of a replication incompetent adenoviral system (BSL-2) reduces risk but requires multiple rounds of plaque purification.
  • Adeno-associated virus: This virus is replication-incompetent and not known to cause disease in humans. If it is prepared using a helper plasmid rather than helper virus, it can often be handled at BSL-1. If prepared using a helper virus, it should be handled at BSL-2. Ethanol cleaning does not inactivate AAV; 10% bleach (0.5% sodium hypochlorite) should be used instead.
  • Lentivirus: Addgene’s lentiviral systems are derived from HIV, but their organization across multiple plasmids and the deletion of many HIV proteins lowers the probability of generating replication-capable virus. These systems are handled at BSL-2/2+.
  • Retrovirus: Retroviruses are classified based on the cell types they infect. For retroviruses that do not infect human cells, BSL-1 may be appropriate; if human cells can be infected, BSL-2/2+ is appropriate.

Vector Characteristics Impacting Biosafety

Envelopes

The viral envelope determines viral tropism, or which cell types a virus can infect. The classes of viral envelopes are:

  • Ecotropic: narrow host range, can infect only one or a small group of species (usually mouse and rat). In this case, biosafety containment procedures might be reduced.
  • Amphotropic: broader host range, usually refers to viruses infecting only mammalian cells.
  • Pantropic: broadest host range; both mammalian and non-mammalian cells can be infected. The VSV-G envelope commonly used to package lentivirus and retrovirus is an example of a pantropic envelope.

Replication-deficient viruses

Most if not all of Addgene’s transfer plasmids are replication-deficient. These plasmids can be used to create virus that is capable of infecting target cells, but cannot produce any new viral particles after the initial infection. This replication deficiency results from the separation of the envelope and packaging proteins from the transfer plasmid. No single plasmid contains all the components necessary to produce viral particles.

Self-inactivation (SIN)

SIN is shorthand for self-inactivating, which is achieved by deleting a large portion of the 3'LTR in retroviral or lentiviral systems. This results in short, inactivated 5' and 3' LTRs and further reduces the possibility of creating replication-competent virus or activating cellular oncogenes. Both 2nd- and 3rd-generation lentiviral vectors can be SIN; however, an internal promoter is required for 2nd generation SIN vectors since the wildtype 5'LTR promoter will no longer be functional.

Transfer Plasmid Insert/Transgene

In addition to the viral vector system, it’s important to consider the properties of the insert/transgene being studied. Inserts that are oncogenic, apoptotic, or toxic present a higher biosafety risk than inserts that do not have these properties, and may warrant more stringent containment procedures.

Viral Genome Percentage

Viral genome percentage is primarily a concern for lentiviral systems derived from HIV. In both 2nd- and 3rd-generation systems, many HIV proteins have been removed. Transfer vectors contain minimal cis-acting HIV components: LTRs, PPT, RRE, and psi packaging signal. Viral components typically total <1.5kb, which should not be more than 30% of the transfer plasmid. As discussed above, many transfer vectors are SIN. Packaging plasmid(s) contain the minimal number of HIV genes required for virus production (3 or 4). Third-generation vectors contain gag, pol, and rev. Second-generation contains these three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is not HIV-derived. In adeno-associated viral systems, only ∼6% of the transfer plasmid (the two ITRs) is viral. Rep and Cap are separated from the transfer plasmid for an extra level of safety.

Additional Resources