EH&S: Biosafety Manual - Recombinant DNA Technology

Recombinant DNA (R-DNA) technology involves combining genetic information from different sources thereby creating genetically modified organisms (GMOs) that may have never existed in nature before. Initially there was concern among molecular biologists that such organisms might have unpredictable and undesirable properties and would represent a biohazard if they escaped from the laboratory. This concern resulted in the famous Asilomar conference held in 1975. At that meeting safety issues were discussed and the first guidelines for R-DNA technology were proposed. More than 25 years have now passed and no adverse incidents associated with this technology have been revealed. This demonstrates that genetic engineering is safe, provided that appropriate safety measures are observed.

R-DNA technology or genetic engineering was first used to clone DNA segments of interest in bacterial hosts in order to produce enough sufficiently pure materials for further studies. More recently, R-DNA molecules have also been used to create genetically modified higher organisms such as transgenic and “knock-out” animals and transgenic plants (see the relevant sections below).

R-DNA technology has already had an enormous impact on biology and medicine and will have an even greater influence in the near future. Now that the nucleotide sequence of the entire human genome is available, tens of thousands of genes of unknown functions will be studied, and R-DNA technology will be one of the means of doing so. Furthermore, gene therapy is expected to become an accepted treatment for certain diseases in the future, and many new vectors for gene transfer will be devised using genetic engineering techniques. Finally, transgenic plants produced by R-DNA technology may play an increasingly important role in modern agriculture.

When considering the use or construction of GMOs, the risk assessment process for work in the laboratory is perhaps even more important than that for work with genetically normal (non-modified) organisms. Whereas the latter are likely to be well characterized with respect to pathogenic properties, the former will be novel, and evaluation of the potential hazards associated with working with such organisms cannot build on experience only.

The risk assessment will identify the biological containment system to be used. The properties of the donor organism, the nature of the DNA sequences that will be transferred, the properties of the recipient organism and the properties of the environment must be evaluated. All of these factors will determine the BSL that is required for the safe handling of the resulting GMO. The following paragraphs provide some background information with respect to these criteria.

Biological expression systems are vectors and host cells that fulfill a number of criteria that make them safe to use. A good example of a biological expression system is plasmid pUC18 (or derivatives thereof), which is frequently used as a cloning vector in combination with Escherichia coli K12 cells. The pUC18 plasmid and its derivatives have been entirely sequenced. More importantly, all genes required for efficient transfer to other bacteria have been deleted from the precursor plasmid pBR322 providing significant containment. E. coli K12 is a strain that lacks the genes known to render some E. coli strains pathogenic. Furthermore, E. coli K12 cannot permanently colonize the gut of healthy humans or animals. Thus, most routine genetic engineering experiments can be performed safely in E. coli K12/pUC18 at BSL 1 provided the inserted foreign DNA sequences do not require a higher BSL (see below).

Risk assessment must consider not only the vector/host system used but also the properties of the DNA to be cloned. In most cases the risk assessment will show that the inserted DNA sequences are unlikely to alter the biological properties of the host organism, but in some cases they may do so, for example, if they are derived from a pathogenic organism. Obviously not all genes of a pathogenic organism contribute to the virulence of the agent. Therefore, insertion of well-characterized DNA sequences that are unlikely to be involved in pathogenicity may not require additional safety measures. However, in cases where these sequences are not characterized, a situation that is typically encountered when a library of genomic DNA of an organism is being established, a higher BSL will be required.

An important consideration is whether the gene product has potential pharmacological activity. Cloning of genes coding for proteins such as toxins may therefore require higher BSLs. Overexpression of gene products from eukaryotic viral vectors can have unexpected consequences when these proteins have pharmacological activity.

Viral vectors are used not only for gene therapy but also for efficient transfer of genes to other cells. Adenovirus vectors have become popular for gene therapy. Such vectors lack certain genes that are required for virus replication and therefore have to be propagated in cell lines that complement the defect. Although such vectors are replication-defective, they should be handled at the same BSL as the parent adenovirus from which they are derived. The reason for this is that the virus stocks may be contaminated with replication-competent viruses, which are generated by rare spontaneous recombination events in the complementing cell line.

Animals carrying foreign genetic information (transgenic animals) should be handled in the containment levels appropriate to the characteristics of the products of the foreign genes. Animals with targeted deletions of specific genes (“knock-out” animals) do not generally present particular biological hazards.

Examples of transgenic animals include animals expressing receptors for viruses normally unable to infect that species. If such animals escaped from the laboratory and transmitted the transgene to the wild animal population, an animal reservoir for that particular virus could theoretically be generated.

This possibility has been discussed for poliovirus and is particularly relevant in the context of poliomyelitis eradication.

Transgenic mice expressing the human poliovirus receptor generated in different laboratories were susceptible to poliovirus infection by various inoculation routes and the resulting disease was clinically and histopathologically similar to human poliomyelitis. However, the mouse model differs from humans in that alimentary tract replication of orally administered poliovirus is either inefficient or does not occur. It is therefore very unlikely that escape of such transgenic mice to the wild would result in the establishment of a new animal reservoir for poliovirus. Nevertheless, this example indicates that for each new line of transgenic animal, detailed studies should be conducted to determine the routes by which the animals can be infected, the inoculum size required for infection and the extent of virus shedding by the infected animals. In addition, all measures should be taken to assure strict containment of receptor transgenic mice.

Transgenic plants expressing genes that confer tolerance to herbicides or resistance to insects are currently a matter of considerable controversy in large parts of the world. The discussions mainly focus on the safety of such plants as food and on the long-term ecological consequences of growing such plants on a large scale, which are not the subjects of this chapter.

Transgenic plants expressing genes of animal or human origin should remain strictly contained within the facility. Such transgenic plants should be handled at BSLs appropriate to the characteristics of the products of the expressed genes.

When creating or handling recombinant organisms, it is essential to perform a detailed risk assessment, which must take into account the nature of the donor, the recipient organism and the environment. In many cases the risk assessment will show that the recombinant organism can be handled at the same BSL as the wild-type recipient. In some instances, however, higher BSLs will be required. This is the case, for example, when ill-defined DNA sequences from a donor organism are transferred, which could potentially increase the virulence of the recipient organism. This situation is typically encountered in random (“shot-gun”) cloning experiments in which genomic DNA libraries are established. Risk assessment is particularly important when creating GMOs expressing proteins with pharmacological activity, such as toxins. It is obvious that such organisms must be handled with caution. Some pharmacologically active proteins are only toxic when expressed at high levels. In this case, the risk assessment becomes very demanding and requires an estimation of the expected expression levels of the protein by a particular recombinant organism and the levels at which a given protein becomes toxic in an organism accidentally exposed to it. The NIH, which established guidelines for work with GMOs, helps scientists classify their work at the appropriate BSL. Risk assessment is thus a dynamic process and has to take into account new developments and the progress of science. It is the responsibility of the scientists involved in genetic engineering to keep up to date on these developments and to respect the guidelines established by the NIH.

1. Recombinant DNA

Experiments involving the generation of R-DNA require registration and approval by the IBC. NIH Guidelines for Research Involving Recombinant DNA Molecules, published by NIH, is the definitive reference for R-DNA research in the U.S. and has been adopted by SDSU's IBC. If the experimental protocol is not covered by the guidelines, contact the BSO at 619-594-2865 for determination of further review. If you have any specific questions about a particular vector/host system not covered by the guidelines, please call the BSO to request review by the IBC.

2. Transgenics

Transgenic Animals

Investigators who create transgenic animals must complete a R-DNA registration document (Appendix D of the BUA Application Form) and submit it to the Graduate and Research Affairs, Division of Research Administration, at ibc@mail.sdsu.edu for IBC approval prior to initiation of experimentation. In addition, IACUC requires that these protocols be approved by its committee prior to initiation of work.

Transgenic Plants

Experiments to genetically engineered plants by R-DNA methods also require registration with the IBC. To prevent release of transgenic plant materials to the environment, the NIH Guidelines for Research Involving Recombinant DNA Molecules published by NIH, provides specific plant biosafety containment recommendations for experiments involving the creation and/or use of genetically engineered plants.


	Business and Financial Affairs > Environmental Health and Safety > Biosafety Program > Biosafety Manual > Contents >
	Environmental Health and Safety SDSU Biosafety Manual San Diego State University BIOHAZARD CONTROL PROGRAM Part VIII: Recombinant DNA Technology A. INTRODUCTION B. BIOLOGICAL EXPRESSION SYSTEMS C. PROPERTIES OF THE DONOR ORGANISM AND CLONED DNA D. VIRAL VECTORS FOR GENE TRANSFER E. TRANSGENIC AND "KNOCK-OUT" ANIMALS F. TRANSGENIC PLANTS G. CONCLUSION H. BIOLOGICAL USE AUTHORIZATION AND REGISTRATION A. INTRODUCTION Recombinant DNA (R-DNA) technology involves combining genetic information from different sources thereby creating genetically modified organisms (GMOs) that may have never existed in nature before. Initially there was concern among molecular biologists that such organisms might have unpredictable and undesirable properties and would represent a biohazard if they escaped from the laboratory. This concern resulted in the famous Asilomar conference held in 1975. At that meeting safety issues were discussed and the first guidelines for R-DNA technology were proposed. More than 25 years have now passed and no adverse incidents associated with this technology have been revealed. This demonstrates that genetic engineering is safe, provided that appropriate safety measures are observed. R-DNA technology or genetic engineering was first used to clone DNA segments of interest in bacterial hosts in order to produce enough sufficiently pure materials for further studies. More recently, R-DNA molecules have also been used to create genetically modified higher organisms such as transgenic and “knock-out” animals and transgenic plants (see the relevant sections below). R-DNA technology has already had an enormous impact on biology and medicine and will have an even greater influence in the near future. Now that the nucleotide sequence of the entire human genome is available, tens of thousands of genes of unknown functions will be studied, and R-DNA technology will be one of the means of doing so. Furthermore, gene therapy is expected to become an accepted treatment for certain diseases in the future, and many new vectors for gene transfer will be devised using genetic engineering techniques. Finally, transgenic plants produced by R-DNA technology may play an increasingly important role in modern agriculture. When considering the use or construction of GMOs, the risk assessment process for work in the laboratory is perhaps even more important than that for work with genetically normal (non-modified) organisms. Whereas the latter are likely to be well characterized with respect to pathogenic properties, the former will be novel, and evaluation of the potential hazards associated with working with such organisms cannot build on experience only. The risk assessment will identify the biological containment system to be used. The properties of the donor organism, the nature of the DNA sequences that will be transferred, the properties of the recipient organism and the properties of the environment must be evaluated. All of these factors will determine the BSL that is required for the safe handling of the resulting GMO. The following paragraphs provide some background information with respect to these criteria. B. BIOLOGICAL EXPRESSION SYSTEMS Biological expression systems are vectors and host cells that fulfill a number of criteria that make them safe to use. A good example of a biological expression system is plasmid pUC18 (or derivatives thereof), which is frequently used as a cloning vector in combination with Escherichia coli K12 cells. The pUC18 plasmid and its derivatives have been entirely sequenced. More importantly, all genes required for efficient transfer to other bacteria have been deleted from the precursor plasmid pBR322 providing significant containment. E. coli K12 is a strain that lacks the genes known to render some E. coli strains pathogenic. Furthermore, E. coli K12 cannot permanently colonize the gut of healthy humans or animals. Thus, most routine genetic engineering experiments can be performed safely in E. coli K12/pUC18 at BSL 1 provided the inserted foreign DNA sequences do not require a higher BSL (see below). C. PROPERTIES OF THE DONOR ORGANISM AND CLONED DNA Risk assessment must consider not only the vector/host system used but also the properties of the DNA to be cloned. In most cases the risk assessment will show that the inserted DNA sequences are unlikely to alter the biological properties of the host organism, but in some cases they may do so, for example, if they are derived from a pathogenic organism. Obviously not all genes of a pathogenic organism contribute to the virulence of the agent. Therefore, insertion of well-characterized DNA sequences that are unlikely to be involved in pathogenicity may not require additional safety measures. However, in cases where these sequences are not characterized, a situation that is typically encountered when a library of genomic DNA of an organism is being established, a higher BSL will be required. An important consideration is whether the gene product has potential pharmacological activity. Cloning of genes coding for proteins such as toxins may therefore require higher BSLs. Overexpression of gene products from eukaryotic viral vectors can have unexpected consequences when these proteins have pharmacological activity. D. VIRAL VECTORS FOR GENE TRANSFER Viral vectors are used not only for gene therapy but also for efficient transfer of genes to other cells. Adenovirus vectors have become popular for gene therapy. Such vectors lack certain genes that are required for virus replication and therefore have to be propagated in cell lines that complement the defect. Although such vectors are replication-defective, they should be handled at the same BSL as the parent adenovirus from which they are derived. The reason for this is that the virus stocks may be contaminated with replication-competent viruses, which are generated by rare spontaneous recombination events in the complementing cell line. E. TRANSGENIC AND “KNOCK-OUT” ANIMALS Animals carrying foreign genetic information (transgenic animals) should be handled in the containment levels appropriate to the characteristics of the products of the foreign genes. Animals with targeted deletions of specific genes (“knock-out” animals) do not generally present particular biological hazards. Examples of transgenic animals include animals expressing receptors for viruses normally unable to infect that species. If such animals escaped from the laboratory and transmitted the transgene to the wild animal population, an animal reservoir for that particular virus could theoretically be generated. This possibility has been discussed for poliovirus and is particularly relevant in the context of poliomyelitis eradication. Transgenic mice expressing the human poliovirus receptor generated in different laboratories were susceptible to poliovirus infection by various inoculation routes and the resulting disease was clinically and histopathologically similar to human poliomyelitis. However, the mouse model differs from humans in that alimentary tract replication of orally administered poliovirus is either inefficient or does not occur. It is therefore very unlikely that escape of such transgenic mice to the wild would result in the establishment of a new animal reservoir for poliovirus. Nevertheless, this example indicates that for each new line of transgenic animal, detailed studies should be conducted to determine the routes by which the animals can be infected, the inoculum size required for infection and the extent of virus shedding by the infected animals. In addition, all measures should be taken to assure strict containment of receptor transgenic mice. F. TRANSGENIC PLANTS Transgenic plants expressing genes that confer tolerance to herbicides or resistance to insects are currently a matter of considerable controversy in large parts of the world. The discussions mainly focus on the safety of such plants as food and on the long-term ecological consequences of growing such plants on a large scale, which are not the subjects of this chapter. Transgenic plants expressing genes of animal or human origin should remain strictly contained within the facility. Such transgenic plants should be handled at BSLs appropriate to the characteristics of the products of the expressed genes. G. CONCLUSION When creating or handling recombinant organisms, it is essential to perform a detailed risk assessment, which must take into account the nature of the donor, the recipient organism and the environment. In many cases the risk assessment will show that the recombinant organism can be handled at the same BSL as the wild-type recipient. In some instances, however, higher BSLs will be required. This is the case, for example, when ill-defined DNA sequences from a donor organism are transferred, which could potentially increase the virulence of the recipient organism. This situation is typically encountered in random (“shot-gun”) cloning experiments in which genomic DNA libraries are established. Risk assessment is particularly important when creating GMOs expressing proteins with pharmacological activity, such as toxins. It is obvious that such organisms must be handled with caution. Some pharmacologically active proteins are only toxic when expressed at high levels. In this case, the risk assessment becomes very demanding and requires an estimation of the expected expression levels of the protein by a particular recombinant organism and the levels at which a given protein becomes toxic in an organism accidentally exposed to it. The NIH, which established guidelines for work with GMOs, helps scientists classify their work at the appropriate BSL. Risk assessment is thus a dynamic process and has to take into account new developments and the progress of science. It is the responsibility of the scientists involved in genetic engineering to keep up to date on these developments and to respect the guidelines established by the NIH. H. BIOLOGICAL USE AUTHORIZATION AND REGISTRATION 1. Recombinant DNA Experiments involving the generation of R-DNA require registration and approval by the IBC. NIH Guidelines for Research Involving Recombinant DNA Molecules, published by NIH, is the definitive reference for R-DNA research in the U.S. and has been adopted by SDSU's IBC. If the experimental protocol is not covered by the guidelines, contact the BSO at 619-594-2865 for determination of further review. If you have any specific questions about a particular vector/host system not covered by the guidelines, please call the BSO to request review by the IBC. 2. Transgenics Transgenic Animals Investigators who create transgenic animals must complete a R-DNA registration document (Appendix D of the BUA Application Form) and submit it to the Graduate and Research Affairs, Division of Research Administration, at ibc@mail.sdsu.edu for IBC approval prior to initiation of experimentation. In addition, IACUC requires that these protocols be approved by its committee prior to initiation of work. Transgenic Plants Experiments to genetically engineered plants by R-DNA methods also require registration with the IBC. To prevent release of transgenic plant materials to the environment, the NIH Guidelines for Research Involving Recombinant DNA Molecules published by NIH, provides specific plant biosafety containment recommendations for experiments involving the creation and/or use of genetically engineered plants.
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Environmental Health and Safety

SDSU Biosafety Manual

Part VIII: Recombinant DNA Technology

Part VIII:
Recombinant DNA Technology