Biosafety Manual PDF - Lawrence Berkeley National Laboratory

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Biosafety Manual IBC-approved version (May 18, 2010) Blood Collection. Source: HHS CDC Office of Health and Safety, Biosafety in the Laboratory presentation (Web accessed May 2010) Appendix H of BMBL states that a risk assessment should be conducted for human and primate cells based on the origin and source of cells or tissues, and such cells should be handled using BL2 practices and containment (see Section 4.4 for further discussion of BLs). While many requirements in the BMBL and OSHA Bloodborne Pathogen Standard are similar to each other, the OSHA standard additionally requires initial and annual BBP training, availability of hepatitis B vaccination at no cost to employees, and a written Exposure Control Plan (ECP). Researchers satisfy documentation requirements for a risk assessment, BL2 containment, and an ECP once they have an approved Biological Use Authorization (BUA). BL2 containment must be used unless the BUA risk assessment indicates that alternative controls are sufficient. BUAs are further discussed in Section 5.1 of this manual and PUB-3000. LBNL work that involves BBP materials will be performed in compliance with the Fed/OSHA Bloodborne Pathogens Standard and BMBL. LBNL’s program for compliance with these standards is integrated into the larger LBNL biosafety program that is described in this manual. 3.3.5 Recombinant Materials, Organisms, and Agents This section defines basic biological terms and processes that are key to understanding recombinant risks and concerns. Genetic material plays a fundamental role in determining the structure and nature of cell substances. It exists in the nucleus, mitochondria, and cytoplasm of a cell or organism, and is capable of self-propagation and genetic variation. The genetic material of a cell can be a gene, a part of a gene, a group of genes, a deoxyribonucleic acid (DNA) molecule, a fragment of DNA, a group of DNA molecules, or the entire genome of an organism. A nucleic acid is a macromolecule composed of chains of monomeric nucleotides. In biochemistry, nucleic acids carry genetic information or form structures within cells. The most common nucleic acids are DNA and ribonucleic acid (RNA). Nucleic acids are universal in living things, as they are found in all cells and viruses. The term genetic recombination is used to describe the process by which the strand of genetic material (usually DNA, but can also be RNA) is broken and then joined to a different DNA molecule to create recombinant genetic material. The NIH Guidelines defines recombinant DNA molecules as molecules constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell or molecules that result from the replication of such molecules. Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version. 24
Biosafety Manual IBC-approved version (May 18, 2010) Vectors are commonly used in genetic engineering to create recombinant materials, organisms, agents, or cells. In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. Such a vector usually does not cause disease itself, but may change the properties and risks associated with the host cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Two common vectors are plasmids and viral vectors. • Plasmid vectors are commonly used to multiply or express particular genes. Many plasmids are commercially available for such uses. Plasmids are DNA segments that are separate from chromosomal DNA and are capable of replicating independently of the chromosomal DNA. In many cases, a plasmid is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms. Plasmids are considered transferable genetic elements, capable of autonomous replication within a suitable host. Plasmid host-to-host transfer requires direct, mechanical transfer by "conjugation" or changes in host gene expression allowing the intentional uptake of the genetic element by "transformation." Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. For example, plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. If these plasmids are inserted into a different host bacterium, the new host may acquire antibiotic resistance or produce toxic protein. • Viral vectors are a viral tool commonly used to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction, and the infected cells are described as transduced. Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells, but once the infection has taken place, it requires a helper virus to produce new virions. Examples of recombinant viral vectors include: • retroviral vectors from retroviruses such as the Moloney murine leukemia virus, • lentiviral vectors from lentiviruses (a subclass of retroviruses) such as HIV, • adenoviral vectors from adenoviruses, and • the adeno-associated virus (AAV). Genetic engineering may also use or create a transgenic organism. A transgenic organism is an organism whose genome has been altered by the transfer of a gene or genes from another species or breed. Examples of transgenic organisms include vertebrates such as mice, plants, and microbes. Work with or the creation of recombinant organisms or specific recombinant genomic materials and nucleic acids may create new risks to humans, animals, plants, or the environment. These potential recombinant risks must be identified and evaluated during the risk assessment process. Examples of genetic modifications that may increase risk include modifications that increase an agent’s pathogenicity or susceptibility to effective treatments (e.g., antibiotics), or increase an organism’s ability to compete in the natural environment. Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version. 25
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