Biological Ethics

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Chapter 19. Manipulation Geneticsby gene transfer

N. López Moratalla and E. Santiago

a) Introduction

The interest awakened in recent years in understanding the mechanism by which genes are expressed and how this expression is regulated has encouraged the development of developmentof techniques that make it possible to transfer information Geneticsfrom one cell to another. These techniques have opened the door to even more ambitious projects aimed at determining and programming the information Geneticsof a particular organism. The areaof biology that encompasses this new knowledge has been given the name "Engineering Genetics", and the term "manipulation Genetics" is often applied to processes aimed at modifying, in some way, the endowment and expression Geneticsof an organism.

Advances in this new field of biology are taking place at a rapid pace, and controversy has often arisen over their possible applications, their legality or illegality in certain cases, and even the possible risk of a biological catastrophe of unsuspected dimensions. Experimentation with the introduction of foreign genes into bacteria and viruses began in the 1970s, and in 1980, the first attempt was made to apply them to humans for therapeutic purposes.

Subsequent initiatives have already led to the creation of "banks" where genes from both prokaryotic and eukaryotic cells are available to researchers for transfer experiments. One such bank contains DNA fragments from Escherichia coli. These DNA fragments incorporated into a plasmid have made it possible to obtain thousands of "clones", i.e. sets of identical copies of the different gene-plasmid hybrids by duplication1 . Another "bank" contains the genes of Drosophila2, and the American Health Administration studied projectto create a human gene bank.

Very soon there was also interest from industry, which saw the possibility of applying these techniques to obtain large-scale gene expression products, such as protein hormones, vaccines, etc.

Spontaneous transfer of genetic material between different species is known to be possible, although it is a rare phenomenon in nature. Genetic material from bacteria or viruses can be incorporated into the genome of a multicellular organism. For example, the plant tumours known as "cockscomb" are induced after infection with the soil bacterium Agrobacterium tumefaciens in a process involving the transfer of a portion of the bacterium's T1 plasmid DNA into plant cells3 ; this portion of bacterial DNA has been found integrated into the plant genome, sometimes even repeated in tandem, and sometimes in reverse. Recombination of discrete DNA fragments from one chromosome with another, of the same or different species - so-called "illegitimate recombination" - appears to be a process involved in cell differentiation in some species. And the transfer of DNA fragments is also thought to have been one of the mechanisms - albeit a rare one - of the evolutionary process.

knowledgeThe application of new techniques to knowledgeof the molecular mechanisms of gene expression and regulation has meant a spectacular and unexpected advance in some of the fundamental problems facing molecular biology, such as the molecular mechanisms of cell differentiation, the alterations that cause cell transformations, or the synthesis of different antibodies. As the French scientist Philippe Kourilsk4 has pointed out, "nobody could have predicted that the possibility of analysing genes would be linked to the ability to act on their expression".

This new knowledge has brought to the forefront the serious responsibility of the scientist to a much deeper understanding of nature than he has ever had before, all the more so as the application of this technology is not without potential risks.

Aware of this responsibility, the initiators of this research subjectimposed a series of restrictions on their workand pushed for the adoption of regulations to control such experimentation; subsequently, all these measures entered a phase of progressive relaxation, as the risks proved to be lower than initially feared.

In response to this statusthe OTA (Office of Technology Assessment) produced a document graduate"Impact of Applied Genetics: Microorganisms, Plants and Animals", which deals with the various aspects of the present and future of engineering Genetics. S. Walton5 , in a commentary on this document, states that this provides congressand the US Administration with extensive material to answer the basic question: "Now that we know how to do it, what are we going to do?

This is also the ethical question. Now that man can exercise such dominion over living beings as he never had before - and logically also over his own body - and can, as has been pointed out, take the course of the evolutionary process into his own hands, what should he do or what should he not do?

b) Engineering Geneticsin bacteria

In 1972, Berg and colleagues produced the first hybrid genome obtained "in vitro" from the animal virus SV-40 and a fragment of the genome of bacteriophage A. This DNA could not be replicated in any animal cell or bacterium, as the replication systems were incomplete6.

In 1973, Cohen obtained the first plasmid vector, PSC101, and in 1974 he introduced the genes encoding penicillin resistance factors into this plasmid7. Subsequently, genes encoding ribosomal RNAs from Xenopus levis introduced into the PSC101 plasmid were cloned into E. coli; these were the first eukaryotic genes transcribed into RNA in bacteria8.

Since then, by means of DNA recombination, it has been possible to isolate and multiply in bacteria a good issueof eukaryotic genes, which has served to acquire information about their location on the chromosome, the mosaic structure and expression of genes of higher organisms and viruses, and to obtain products of interest at an industrial level.

Copies of these genes are now available from higher organisms - for example, from various human and rat globulins, rat immunoglobulins, chicken ovalbumin, lysozyme, hormones such as somatostatin, insulin, human growth hormone and interferon, etc. - that can be expressed in bacteria.

In the not too distant future, DNA recombination is expected to have a direct impact on fields as diverse as pharmaceuticals, environmental protection and agriculture. New antibiotics will be developed using these techniques, and hormones, enzymes and vaccines will become easier to obtain. For example, the use of cellulose, an inexpensive substrate, in fermentation processes through the introduction of the cellulose gene will be attempted to produce synthetic rubber, pesticides, etc. These techniques could also be used to obtain micro-organisms to transform inedible biomass into food, or to help protect against various forms of environmental pollution. It will also be possible to "influence" multiple biological processes in micro-organisms, such as the developmentof metabolic pathways that require less energy, enzymes with more suitable kinetic properties, not subject to control by retro-inhibition, etc.

The positive aspect of these technologies is clear: they have helped and will continue to help clarify aspects of the endowment Geneticsof the different species and their expression, of undeniable value. At the same time, they allow a mastery of living beings which, oriented towards the service of man, is also a valuable scientific achievement. This orientation requires that ethics be present in scientists' decisions, since any technical manipulation of nature always has an ambivalent character.

Regulating the use of recombinant technologies

The ethical evaluationof these technical applications has, from the outset, been raised in a climate of intense controversy and has focused primarily on the aspect of potential risk.

These dataare carried out between 1974 and 1980 and can be divided into three stages.

1st Stage: The intervention of the Commission of the National Academy of Sciences of the United States on DNA Recombination.

In 1974, the US National Academy considered the possibility that widespread or unreasonable use of these techniques, because of the potential biohazards involved, could have irremediable consequences and asked Paul Berg to form an advisory committee to study the issue. For years, Berg had been concerned about the potential dangers of certain types of research. In the spring of 1974, Berg assembled a groupof researchers, some of whom had worked with him, such as Stanley, Cohen and Boyer, on DNA recombination techniques.

In a reportpublished in June and in a letter addressed to the main specialised journals, the members of the commission expressed "their concern about the possible consequences of the indiscriminate application of the techniques"9 of engineering Geneticsand formally requested all researchers to join them by voluntarily renouncing certain experiments. This letter presents a first typification of potentially dangerous experiments.

One of the main concerns of the Academy of Sciences Commission was the inability of scientists to specify and narrow down the dangers of certain experiments before carrying them out, in contrast to what happened in other potentially dangerous fields of research, such as those programs of studycarried out with pathogenic bacteria and viruses, radioactive isotopes or toxic products, where very strict safety measures have always been observed. However, due to the novelty of the methods of microbial Geneticsmanipulation, no rules had yet been established for this subject. There was a possibility that potentially dangerous experiments could be carried out without any safety measures. The Commission therefore recommended the withdrawalof some experiments until the hazards were more precisely identified, i.e. until it could be determined whether or not the workwould be carried out safely and appropriate precautions could be taken.

high schoolThe Commission proposed that an international meetingbe held in early 1975 to consider these problems more broadly, and that the National Institutes of Health (NIH) be established as the official watchdog for experimental programming in this field and that a code of ethics for researchers working in engineering Genetics be drafted as soon as possible.

Stage 2 - Asilomar's International congresson Molecular Recombination of DNA.

The congresstook place in February at the Asilomar Conference Center near Pacific Grove, California. It was attended by 86 American biologists and 53 researchers from 16 other countries, who spent three and a half days discussing progress in the field of engineering Geneticsand developing safeguards that would allow novel heritable traits to be introduced into both bacteria and viruses without unnecessary risk. Invited guests included professionals from related fields of law, as well as representatives of institutions providing funding for scientific research. The meetings were open to the press and extensive information was provided.

The committeeorganiser of the congressdrafted a report10 which was submitted to the Academy of Sciences and Cby its Executive committeeon 20 May 1975. The same committeeof Asilomar gave the scientific press a summaryof such a report(cf. Annex), which already represents a systematisation of the generic indications given by P. Berg the previous year. Rules for experimentation were established according to fundamental criteria; the risks of dissemination of the biological combinations produced and the molecules used were taken into account.

The differences of opinion expressed on congressand the problems discussed there were given great prominence by science editors and journalists. From then on, an alarmist atmosphere arose in public opinion. The disorientation was reflected by an abundance of letters to newspapers and the appearance of sensationalist articles in the press11 12.

A position of ethical relativism became generalised, according to which it seems that ethical criteria should be born from a sociological surveyand base their validity on the free and majority capture of public opinion, lacking serious scientific information and a highly specialised deontological preparation and trainingon subjects such as this one.

3rd Stage: Exceeding the established deontological regulations.

Following numerous debates between scientists and public opinion, governments began to take an interest in topicand legislation emerged in the United States, France and England13 . high schoolMore or less rigid legislation, mainly derived from the first code of ethics published in 1976 by the National Institutes of Health (NIH)14, differed essentially in the different coercive power of the authority that enacted them and which became managerresponsible for controlling the application of the laws themselves.

Numerous factors have contributed to the fact that in a few years these regulations have been greatly relaxed. On the one hand, because the feared risk of obtaining pathogenic organisms has resultbeen much reduced, while the possibility of use for human gene therapy and new forms of eugenics has attracted media attention. On the other hand, experiments that had been refused licenceto be carried out in one country have been carried out in another. Another decisive factor was that the committees responsible for the approval of the proposed experiments declared themselves unable to judge them, because they could not foresee the possible implications15 16.

evaluationethics

The codes of ethics on DNA recombination experiments are a financial aidin the determination, not always easy, of the possible negative effects and of the necessary means to ensure the developmentof the experiments, following reasonable safety and isolation standards.

In 1976, the NIH (high schoolNational Institutes of Health of the United States) proposed a code of ethics that presupposes the moral legitimacy of experiments. It is not possible to seek in this code what it does not claim to provide, i.e. ethical evaluations of experiments. In fact, it does not address the topicof the applications of these techniques to humans, nor the problem of using experiments for destructive purposes. It limits itself to considering the dangers of the effects of DNA recombinations in viruses, bacteria and eukaryotic cells; its considerations are therefore partial and insufficient, as they do not cover the entire areaof engineering Genetics, but they are and imply an expression of responsibility on the part of scientists by promotethat certain competent institutions delimit the sphere within which their activity can move.

For an ethical evaluationit is also necessary to take other factors into account. On the one hand, the physical object of the experiment; it is not the same for plants, animals and micro-organisms as it is for humans. On the other hand, the intended purpose. Techniques alone do not determine the morality of an experiment; it becomes lawful or unlawful, good or bad ethically, also according to the intended purpose of researcher, and the accidental side effects that derive from it.

With regard to engineering Geneticsin the different species - with the exception of man - it must be affirmed that it must always be ordered to serve man, directly or indirectly17. The researchercannot, therefore, have the intention of producing damage. The limits of the notions of service and harm to man and the criteria relating to their distinctions are given by the criteria and immediate conclusions of natural law, principles that constitute the so-called rights of the person. Engineering experiments Genetics- transformation, transduction, cell fusion, etc. - would not be licit when the researcherintends to produce pathogenic agents for purposes that directly or indirectly threaten the physical and psychological integrity of man; the goalsought cannot be "biological warfare" (cf. chapter 25).

As far as the effect of engineering experiments Genetics is concerned, it can be good or indifferent. Man has control plenary session of the Executive Council, although not absolute, over the inert world and over living beings, and can even modify the constitution Geneticsof organisms.

To a large extent, the background to the debates - to which we alluded earlier - and the fear aroused is due to the realisation that with these techniques, the power that man in the last quarter of the 20th century acquired over the living world was unprecedented. The extreme positions are opposed, in the midst of fierce polemics, without being able to provide a solution. The proposalof a complete and radical withdrawalof experiments in this field comes from the side of ecological ideologies; if nature has absolute primacy over reason, the natural constitutes the only criterion that marks the limits beyond which applied science should not go. The opposite position defends the elimination of barriers that prevent doing everything that is in fact possible; it is based on the assumption that all action, in general, is morally indifferent, and the decision is left to the sole responsibility of the experimenters and, therefore, something in particular is good or bad according to the conscience of the decision-maker. It is a mere consequentialism in which only the good intention of the doer matters and not the act itself. But with a generic desire to do good as the ultimate end, no judgement is made about the means, not even about the hierarchy of partial ends, nor about responsibility for side effects that are not easily predictable.

There is an underlying problem here about man's dominion over nature. The "dark" awareness of the seriousness of manipulation Geneticsis due to the implicit conviction that the world has an order of its own and that such manipulations can trigger an irreversible disturbance in the order of the cosmos. This is clearly not the case in other human interference, even in the biological world, because in normal interference, the order of the world can "digest" such interventions without changing it. The fact that there are natural gene transfers is no guarantee that with other manipulations the ecological balance will not be lost. Thus the criterion that often implicitly informs these codes of ethics is that of avoiding loss of control; this criterion may be basically valid, though not in itself, but as an expression of respect for the order of the universe. This is an ethical criterion on the researchitself and is a criterion prior to the concrete purpose of the researcher.

The other factor involved in determining the lawfulness of their conduct is the secondary and accidental effect or consequences. If there are accidental bad effects, there must be a serious cause for conducting such experiments that can justify the unintended accidental effects and that is at the same time proportionate to the seriousness of the effects. Ignorance of possible bad side effects may be wilful and therefore culpable for lack of diligence in the preparation of the experimental plan at work. This requires a prior bibliographic knowledgeof the background of the experiment and the problems it raises. The seriousness of the scientific workrequires, in fact, accurate and prior initial and immediate documentation, and, in relation to the plan to be developed, a evaluationof the technical means available to carry out the experiment, without harming the rules of natural ethics relating to the right to physical, psychological and moral integrity of researcher, of his team workand of others.

c) Engineering Geneticsin plants and animals

Plants

The high cost of soil fertilisation with nitrogen fertilisers has led to increasing attention being paid to research on atmospheric nitrogen fixation, with the possibility of modifying and transferring the genes encoding the complex enzymatic machinery - 17 proteins - required for this process using engineering techniques dominating this work Genetics. The fundamental aim of goalis to make crops fix nitrogen. On the one hand, fixer species such as Rhizobium, a legume symbiont, will produce nodules on a non-legume plant; on the other hand, bacteria such as Azotobacter vinelandii, which has no symbiotic relationship with any plant, will attach to cereal roots to provide them with nitrogen that has already been reduced. An even more ambitious projectis to achieve gene transfer from NIF (Taxpayer Identification Number)to the plant18 . Such genes have been transferred into a nitrogen-fixing bacterium, and more recently into a yeast. But what has not yet been achieved is the expression of these genes NIF (Taxpayer Identification Number)in eukaryotes.

To achieve the transfer of the genes NIF (Taxpayer Identification Number)into a plant, it is possible to transfer them into plant cells in culture and direct, through the use of plant hormones, the differentiation of these cells into a plant. It is also possible to transfer the genes to the seed, so that the genes are then expressed in the plant19. The T1 plasmid from the bacterium Agrobacterium tumefaciens, which is able to enter a wide variety of phanerogams and insert itself into the DNA of their genomes, can be used as a vector for these genes. The DNA of this plasmid is maintained in all cells infected with the plasmid. Gene transfer, using cauliflower mosaic virus as a vector, is also being studied.

So far, genes from Rhizobium have been transferred into A. vinelandii, resulting in its attachment to maize18. And Hooykaas and co-workers20 have shown that the Rhizobium trifolii Sym plasmid carrying host specificity, which controls the steps required for nodulation and nitrogen fixation in clover roots, is capable of expression in other Rhizobium species and in Agrobacterium tumefaciens.

Although the technical difficulties are great, it is not unreasonable to think that it will soon be possible to introduce resistance genes against pathogens and insects into plants. Since pesticides and insecticides are not only expensive, but also pollute the environment, great efforts are currently being made to provide plants with their own defence systems. For example, a DNA hybrid between an Agrobacterium promoter and toxins from Bacillus thuringiensis provides the plant with defence against insects21 . On the other hand, plants produce an inhibitor against insect proteases whose synthesis is induced by a factor in response to mechanical injury caused by the insect. The genes for these protein inhibitors have been isolated and transferred to other plants; for example, the potato gene has been transferred to tobacco and induction of the gene has been observed if the 3'untranslatable region of the potato gene is linked to it22 .

The use of the A. tumefaciens T1 plasmid has been shown to be very effective for gene transfer in dicots. For others, direct gene transfer to protoplasts in culture has been used, leaving the whole plant to develop later23. In cereals, genes encoding kanamycin resistance have been introduced by DNA injection24.

Animals

Until very recently, the idea of being able to modify the genome of an entire organism seemed far away. Since 1980, however, the statushas changed with the expression of foreign genes in the mouse. But the possibility of introducing a gene "at will", placing it correctly on the chromosome and passing it on faithfully to the offspring is not without its difficulties. Various methods have been used to carry out this gene transfer: direct injection into the zygote or embryos, and transplantation of previously corrected cells.

Direct injection into the zygote(s) - By microinjection, the introduction of genes into the nucleus of a zygote can be achieved at the time of in vitro fertilisation. The new information Geneticscan be maintained throughout the embryo development.

Earlier experiments by Mertz and Gordon25 had already achieved the transcription of histone genes from "Drosophila" into the "Xenopus" oocyte. Gordon etal26 have introduced foreign genes, the viral thymidine kinase gene, a bacterial plasmid fragment and SV40 DNA, into mouse zygotes. Of the 180 zygotes that developed, three carried these foreign genes in their genome and in one of them, the grafted DNA was not integrated into the chromosomes, which could mean either that it replicated as an independent plasmid or that it was an extrachromosomal copy of an integrated portion. Jaenisch and Mintz27 have injected SV40 DNA into mouse blastocysts and the animals developed after implantation of these blastocysts in the maternal uterus retained these DNAs into adulthood.

In 1985, the expression of genes introduced by microinjection into the pronucleus or nucleus of the fertilised egg was achieved in rabbits, sheep and pigs28 . There are many advantages that these techniques applied to domestic animals can have; some obvious ones are, for example, increased reproductive efficiency, weight gain, increased resistance to disease, change in skin texture. Amplification of the gene encoding growth hormone had a dramatic effect in rats, which, however, has not occurred in pigs or rabbits29.

Modified cell transport - Cline's group30 31 has succeeded in inserting, by transformation, genes into mouse bone marrow cells in culture. The transferred genes correspond to dihydrofolate reductase, which confers resistance to methotrexate, a drug that was applied once the cells were transplanted, with the idea of selectively favouring their proliferation.

Using a similar system, Pellicer etal32 have introduced the gene encoding herpes simplex thymidine kinase and human globin into mouse teratocarcinoma cells, and once the cells were transformed in culture, they were transplanted into the animals.

The successful transplantation of teratocarcinoma cells into embryos33 opens up the possibility of using this route to introduce new genes, without the need for the administration of a drug to exert selective pressure on their proliferation, as the teratocarcinoma cells, in this embryonic environment, lose their malignancy and develop normally. There is a difficulty, however, and that is that when these cells are grown in culture, they acquire an abnormal number of chromosomes issue.

Use of retroviral vectors - Robertson et al.34 succeeded in 1986 in introducing exogenous genes using retroviral vectors into stem cells and have shown that these modified cells contribute extensively to somatic cell and formative lineage traits in chimeric mice. This system makes it possible to use these techniques to modify and select potentially germline cells, which can then be transferred with predetermined genetic changes.

evaluationethics

For the time being, the technical difficulties involved in applying DNA recombination techniques to animals mean that the possibilities of revolutionising animal husbandry35 by modifying the breeding Geneticsof animals for human use are not only slim and remote, but also practically limited to the improvements that could result from the introduction of a single gene.

From an ethical point of view, the application of these techniques to plants and animals does not present any particular problems. To a large extent, this experimentation does not differ much from that practised since ancient times by breeders, plant breeders and livestock breeders, carrying out a strong selection directed towards their own interests; it does differ in terms of the possibility of incorporating new traits into a species. Similar to what we have already seen in the case of micro-organisms, the modification of heritage is an action which, although it implies a drastic intervention in living beings, is licit if the aim is to improve the species in the service of man, or as a means of researching and acquiring new knowledge. On the other hand, these applications do not present the dangers that could arise with micro-organisms, where there is a risk of losing control of new pathogenic strains, etc.

The lectureof Rambouillet and other meetings

In April 1985, the 1st International Bioethics colloquiumwas held to evaluate technology from an ethical point of view Genetics. The following recommendations were made:

1st Genetics : To encourage the basic in transfer techniques in plants and animals, and, in general, on what refers to the mechanisms of vital processes. research

2nd: Supervise the developmentof the use of technological-genetic processes in agriculture, especially with regard to their ecological risks, minimising them as is done in the pharmaceutical sector.

3rd: Public and private institutions in all countries should have access to the published databanks, as "secrecy" in this field contravenes the public interest.

4th: Do not forget in the researchof the human embryo its moral condition as a potential human person.

Subsequently, a wide variety of meetings, symposia, etc. have been held to discuss the possibilities, repercussions and risks of DNA recombination, including its application to humans. García Prada36 points out: "From their position and with their perspective, scientists argue that the progress of science is unstoppable, that there is no "non plus ultra" at research. Here precisely lies the decisive point core topicin the excessive scientific-technical pretension of technologically-genetically correcting human biological imperfection by manipulating its mass Genetics, in order to achieve the desired perfection Degree.

This was initially proposed with enthusiasm (J. Muller, J. Lederberg) at the CIBA symposium in 1962 and is still heard occasionally as an echo or foreshadowing of A. Huxley's visions in "Brave New World". If society consents to this, the door would be opened to the "totalitarianism of science" (E. Benda) and the scientist would usurp the functions of the Creator and of evolution (G. Fulgraff, former German Health Minister director ). What a New York Times editorialist called "the remaking of the species" is at stake. It is therefore necessary to discuss the issue further, because its tremendous concomitant dimension is not only the abuse but also the use of this technology itself, and not only the extreme case of genome recombination with its irreversible and unforeseeable consequences for the offspring.

d) Biotechnology and environment

Since 1986, the release of modified organisms into the environment has once again given rise to the need for legal controls37.

A controversy has arisen over the use of an altered bacterium - a new strain of Pseudomonas syringue to protect strawberries from freezing damage; this altered bacterium is missing the fragment of the genome that encodes the protein that produces the ice core and is managerfrom freezing damage to the plant. The new strain protects by competing with the wild-type strain for position on the leaves38.

In England, an experiment on the release of a virus used for biological pest control39 has been carried out at Cwith strong environmentalists civil service examination.

Another controversy has started with the use of a preventive vaccine for pseudorabies caused by a herpesvirus that primarily affects pigs. The mutated vector virus lacks the thymidine kinase gene managerfor the pathogenic effect40.

An altered strain has also been obtained that produces a beer leavein calories. This strain of Saccharomyces uvarum contains a gene from the mould Aspergillus niger that encodes a glucoamylase. Although during the five years of this work it was pasteurised in order to kill the bacteria, unpasteurised beer will subsequently be served in pubs and thus contain live micro-organisms that could be released into the environment41.

It is obvious that in the use of agents such as these, even if they are not themselves pathogenic, extreme caution must be exercised; and it does not seem lawful to use them on a large scale without first checking that they do not produce dangerous effects through the possible integration of foreign genes into other genomes.

Notes

(1) CLARKEL, L. and CARBON, J. "A colony bank containing synthetic hybrid plasmids representative of the entire E. coli". Cell, 9, 99, 1967.

(2) WENSKI, P.C., FRIMEGAN, D.T., DONELSON, J.E. and HOGUESS, D.S. "A system for mapping DNA sequences in the chromosomes of Drosphila melanogaster. Cell, 3, 315-325, 1974.

(3) ZAMBRYSKI, P., HOLSTER, M., KRUGER, K., DEPICKER, A., SCHELL, J., MONTAGU, M.V. and GOODMAN, H.M. 'Tumor DNA structure in plant cells transformed by A. tumefaciens'. Science, 209, 1385-1391, 1980.

(4) KOURILSK, P. "La génie génétique". La Recherche, 110, 390-402, 1980.

(5) WALTON, S. "OTA weighs the impact of genetic engineering". Bioscience, 31, 198-199, 1981.

(6) JACKSON, D.A., SYMONS, R.H. and BERG, P. "Biochemical method for inserting new genetic information in vitro of DNA of simian virus 40". Natl. Acad. Sci. USA, 69, 2904-2909, 1972.

(7) CHANG, A.C.Y. and COHEN, S.N. 'Genome construction between bacterial species in vitro: Replication and expression of Staphylococcus plasmid genes in Escherichia coli'. Proc. Natl. Acad. Sci. USA, 71, 1030-1034, 1974.

(8) MORROW, J.F., COHEN, S.N., CHANG, A.C.Y., BOYER, H.W., GOODMAN, B.M. and HELLING, R.B. "Replication and transcription of eukaryotic DNA in Escherichia coli". Proc. Natl. Acad. Sci. USA, 71, 1743-1747, 1974.

(9) BERG, A.A. "Potential biohazards of recombinant DNA molecules". Science, 185, 303, 1974.

(10) BERG, P. et al. "Asilomar conference on DNA recombinant molecules". Nature, 255, 444, 1975.

(11) GROUPE INFORMATION BIOLOGIE. "Manipulations génétiques pour un débat public. La Recherche, 6, 692, 1975.

(12) PEREIRA DE SILVA, L.H. "Manipulations génétiques contre l'obscurantisme". La Recherche, 5, 790, 1975.

(13) BLANC, M. "Le vrai visage des manipulations génétiques". La Recherche, 97, 188-191, 1979.

(14) NATIONAL INSTITUTE OF HEALTH. "Guidelines for research involving Recombinant DNA Molecules. NIH. Washington, 1976.

(15) "Will the DNA guidelines wither away? Nature, 287, 473, 1980. "Scares about genetic manipulation". Nature, 287, 96, 1980.

(16) DICKSON, D. "DNA guidelines. More relaxation". Nature, 287, 1980.

(17) LEJEUNE, J. "Relazione all'Accademia francese di science morali politiche. Alle origini della vite. L'oumo nasce uomo". Studi Cattolici, 155, 19, 1979.

(18) BRILL, W.J. "Agricultural Microbiology". researchand Science, 62, 118-120, 1981.

(19) CARLSON, P.S. 'The use of protoplasts for genetic research'. Proc. Natl. Acad. Sci. USA, 70, 598, 1973.

(20) HOOYKAAS, P.J.J., VAN BRUSSEL, A.A.N., DEN DULK-RAS, H., VAN SLOGTEREN, G.M.S. and SCHILPEROORT, T.A. "Sym plasmid of Rhizobium trifolii expressed in different rhizobial species and Agrobacterium tumefaciens". Nature, 291, 351-353, 1981.

(21) VEACK, M. "Transgenic plants protected from insect attack". Nature, 328, 33-37, 1987.

(22) SHIELDS, R. "Towards insect-resistant plants" Nature 328, 12-13. 1987

(23) Examples are tobacco (BLOCK, P. et al., EMBO 3, 1681-89, 1984); petunia (LAMPA, G. et al. Nature 316, 750-757, 1985); and tomato (HORSCH, R.B. Science 227, 1229-31. 1985).

(24) DE LA PEÑA, A., LIRS, M., SCHELL, J. "Transgenic rye plants obtained by injecting DNA into young floral tillers". Nature, 325, 274-276, 1987.

(25) MERTZ, J.E. and GORDON, J.B. "Purified DNAs are transcribed after microinjection into Xenopus oocytes". Proc. Natl. Acad. Sci. USA, 74, 1502-1506, 1977.

(26) GORDON, J.W., SCANGOS, G.A., PLOTKIN, D.J., BARBOSA, J.S. and RUDDLE, F.H. "Genetic transformation of mouse embryos by microinjection of purified DNA". Proc. Natl. Acad. Sci. USA, 78, 1981.

(27) JAENISCH, R. and MINTZ, B. "Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA". Proc. Natl. Acad. Sci. USA, 71, 1250-1254, 1974.

(28) HAMMER R.E. et al. "Production of transgenic rabbits, sheep and pig by microinjection". Nature 315, 680-683, 1985.

(29) PALMITER, R. D., BRINSTER, R. L., HAMMER, R. E., TRUMBAUER, M. E., ROSENFELD, M. G., BIRNBERG, N. C. and EVANS, R. M. "Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature, 300, 611-615, 1982.

(30) MERCOLA, K.E., STANG, H.D., BORWNE, J., SALSER, W., and CLINE, M.Y. "Insertion of a new gene of viral origin into bone marrow cells of mice". Science, 208, 1033-1035, 1980.

(31) CLINE, M.J., STANG, H., MERCOLA, K., MORSE, L., RUPRECHTER BROWNE, J. and SAISEL, W. "Gene transfer in intact animals". Nature, 248, 422-425, 1980.

(32) PELLICER, A., WAGNER, E.F., ELKAREH, A., DEWEY, M.J., RUESSER, A.J., SILVERSTEIN, S., AXEL, R. and MINTZ, B. "Introduction of a viral thymidine kinase gene and the human -globin gene into developmentally multipotential mouse teratocarcinoma cells". Proc. Natl. Acad. Sci. USA, 77, 2098, 1980.

(33) MINTZ, B. "Genetic mosaicism in adult mice of quadriparental lineage". Science, 148, 1232-1333, 1965.

(34) ROBERTSON E, BRADLEY A, KUEMN M and EVANS M "Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector" Nature, 323, 445-448, 1986.

(35) TANGLEY L. "Biotechnology on the farm". Bioscience, 36, 590-593. 1986.

(36) GARCIA PRADA, O. "Biogenetics and responsibility". programs of studyFilosóficos, 98, 64-102, 1986.

(37) The journal Nature, in volume 326 of 1987, devotes several articles to this topic: pp. 537 and 819.

(38) PALCA, J. "Frost damage tests blocked" Nature 319, 254. 1986; JUKES, T.N. "Frost resistance and Pseudomonas". Nature, 319, 617, 1986.

(39) NEWMARK P "Approval for first British virus release experiment". Nature, 320, 2, 1986.

(40) BEARDSLEY, T. "USDA goes too public too quickly". Nature, 320, 473, 1986.

(41) PALCA, J. "Living outside regulation". Nature 324, 202, 1986.

 

Annex: The lectureof Asilomar

At a meetingheld in California from 24-27 February, an international groupof scientists decided that strict controls should be placed on the use of the experimental technique allowing the transplantation of genes from one organism to another. This statement drafted by the committeeOrganising lectureis the summaryof a reportsubmitted to the Life Sciences Assembly of the National Academy of Sciences and Cby its executive committeeon 20 May 1975.

This meetingwas organised to review the scientific progress at researchon recombinant DNA molecules and to discuss appropriate ways to address the potential biological risks of this work. The impressive scientific advances that have already been made in this field and in these techniques are of great importance in moving towards an understanding of fundamental biochemical processes in prokaryotic and eukaryotic cells. The use of DNA recombination methodology promises to revolutionise the internshipof molecular biology. Although no application of the new techniques has so far taken place, there is every reason to believe that they will be of great use internshipin the future.

The attention of the participants to the meetingwas particularly directed to the question of whether the suspension provisional of certain aspects of researchon this area, imposed by the committeefor recombination of DNA molecules of the US National Academy of Sciences, in the letter published in July 1974, should be lifted; and if so, how the scientific workcould be undertaken with minimal risks to the staffof laboratorydedicated to this subjectof work, to the general public, and to the animals and plants that share our ecosystem.

New techniques that allow the combination of information Geneticsbetween organisms that are very different from each other place us in a areaof biology with many questions. Even today, the fact that we have limited researchin this field makes evaluationof the possible risks extremely difficult. It is this ignorance that has prompted us to decide that it would be prudent to take considerable precautions in conducting this research. However, participants at lectureagreed that most of the workon the construction of recombinant DNA molecules should continue as long as appropriate safety measures are employed, mainly in terms of adequate biological and physical barriers to contain the newly created organisms. The criteria for protection should be made even more stringent at the outset and modified as methodology improves and a more accurate evaluationof the risks becomes available. It was also agreed that there are certain experiments where the potential risks are so high that they should not be conducted given the current limited means. In a longer term deadlineproblems may arise in the large-scale application of this methodology in industry, medicine and agriculture. But it was also recognised that future researchand experience may show that many of the potential risks are less serious and less likely than we now suspect.

Principles guiding the recommendations and conclusions

Although our statements about the risks that may be involved in each of the different pathways of research, on the recombination of DNA molecules may differ, few, if any, people believe that this methodology is entirely risk-free. The principles of prudence in dealing with these potential risks are:

That the use of appropriate barriers is considered essential on the experimental project, and that this protection and isolation is proportionate to the potential risk. Consequently, alongside a scale of risk, there should be a corresponding scale of protection and isolation. Risk estimation will be difficult and intuitive at first, but will improve as new knowledge is acquired; at each stage it will be necessary to check that protection and isolation are appropriate to the possible risk. It seems logical that experiments requiring larger-scale operations will involve much more serious risks than those involved in small-scale experiments, and therefore require more rigorous protection and isolation procedures. The use of clonal vehicles or vectors (plasmids, phages) and bacterial hosts, with a restricted ability to multiply outside laboratory, would reduce the risks of a given experiment. Therefore, ways to match isolation levels to potential risks may change over time, especially as protection and isolation techniques improve. The means of assessing and balancing risks with appropriate levels of isolation and protection should be reviewed periodically. It is to be expected that, through both formal and informal national and international information channels, the way in which potential biological risks and protection are addressed will be reasonably consistent.

Isolation of potentially harmful agents can be achieved in several ways. The most important contribution to limiting the spread of recombinant DNA is the use of biological barriers. These barriers are of two types: 1) Harmful bacterial hosts unable to survive in a natural environment; 2) Non-transmissible and equally harmful vectors (plasmids, bacteriophages or other viruses) able to develop only in specific hosts. Physical isolation, exemplified by the use of hoods or, where possible, limited access to negative pressure laboratories, provides an additional safety factor. Of particular importance is strict and stringent microbiological internship, which can greatly limit the escape of organisms from the experimental environment, and thus increase the safety of the operation. Therefore, the Educationand trainingof all staffinvolved in the experiments is essential for the effectiveness of all isolation measures.

On internship, these different means of isolation will complement each other and substantial advances in the developmentof bacterial hosts and vectors may allow modifications to the complementary physical isolation requirements.

Strict physical isolation and rigorous laboratoryprocedures can reduce, but not eliminate, the possibility of potentially dangerous agents. Therefore, researchers basing their workon inactivated hosts and vectors, as an additional safeguard, should rigorously test the effectiveness of these agents before accepting their validity as biological barriers.

Recommendations for matching types of isolation to types of experiments

No classification of experiments in terms of risk, and no set of isolation procedures can foresee all possible situations. Given our current doubts about risks, the parameters proposed here are broadly conceived and intended to provide a guidelineprovisional for researchers and centres involved in researchrecombinant DNA. However, it is up to each researcherindividual to decide whether, in a particular case, special circumstances justify a higher level of isolation than suggested here.

subjectcontainment

1. Minimal risk: This isolation subjectis suitable for those experiments where biohazards can be accurately assessed and can be expected to be minimal. Such isolation can be achieved by following the procedures recommended for clinical microbiology laboratories. These measures focus primarily on not drinking, eating, or smoking in the laboratory, the use of gowns in the areaof work, the use of cotton-capped pipettes or preferably mechanical pipettes, and prompt disinfection of contaminated materials.

2. Low risk: This level of isolation is appropriate for experiments that generate novel biotypes, except where available information indicates that the recombinant DNA would not appreciably alter the ecological behaviour of the recipient species, significantly increase its pathogenicity, or preclude effective treatment of the resulting potential infection. Essential features of this isolation (in addition to the minimum procedures, mentioned above) are the prohibition of the use of mouth pipettes, limited access to stafffrom laboratory, biological safety cabinets for procedures that may produce aerosols (e.g. mixing and sonication). Although existing vectors can be used at this level of isolation for low-risk work, safer vectors and hosts should be adopted where available.

3. Moderate risk: Such isolation measures are appropriate for experiments where there is a likelihood of generating an agent with significant potential, in terms of pathogenicity or ecological destruction. The main features of this safety level, in addition to the two preceding classes, are that transfer operations should be performed in biosafety cabinets (e.g. laminar flow hoods), gloves should be worn during handling of infectious materials, vacuum lines should be protected by filters, and negative pressure should be maintained in limited access laboratories. However, experiments offering moderate risk should be performed only with vectors and hosts that have a very low capacity to multiply outside the laboratory.

4. High risk: This safety level is intended for experiments where the potential for ecological destruction or pathogenicity of the modified organism may be severe and therefore presents a very serious hazard to the staffof laboratoryor to the public. The main features of this measurement subjectare the same as those used in the handling of extraordinarily infectious microbiological agents, and consist of measures to isolate the areaof workfrom other areas, by means of air locks, a negative pressure environment, the requirement for changes of clothing, a shower for staffand laboratories equipped with systems for the inactivation or elimination of biological agents that may be contained in the foul air and in liquid or solid waste. All persons occupying these areas should wear protective gowns and shower at each exit from the special isolation area. Biological agents should only be handled in biological safety cabinets, where the foul air is incinerated or passed through special filters. Isolation for the high-risk workincludes, in addition to the physical and procedurecharacteristics described above, the use of rigorously tested vectors and hosts whose developmentcan be confined to laboratory.

Types of experiments

Accurate estimates of the risks associated with different types of experiments are difficult to obtain, due to our ignorance of the likelihood of the anticipated risks manifesting themselves. However, experiments involving the construction and propagation of recombinant DNA molecules from: 1) prokaryotes, bacteriophages and other plasmids; 2) eukaryotes, have been classified as minimal, low, moderate and high risk to guide researchers in their choice of appropriate protection. These designations should be considered as provisional assessments, which will need to be reviewed in the light of future experience.

The recombinant DNA molecules themselves, as distinct from the carrier cells, may be infectious to bacteria or higher organisms. DNA preparations in these experiments, especially in large quantities, should be chemically inactivated before disposal.

1. Prokaryotes, bacteriophages and bacterial plasmids: Where the construction of recombinant DNA molecules and their propagation involves prokaryotic agents known to naturally exchange information Genetics, experiments can be performed with minimal risk safety measures. Whenever experiments give rise to suspicion of potential risk, more stringent protection should be ensured.

Experiments involving the creation and propagation of recombinant DNA molecules from DNA of species that do not ordinarily exchange information Genetics, generate novel biotypes. Since such experiments may offer higher risks than those related to the original organisms, they should at least be done with the safety measures recommended for low-risk experiments. If the experiments involve pathogenic organisms, or genetic determinants that may increase the pathogenicity of the carrier species, or if the transferred DNA may confer on the carrier organisms new metabolic activities not native to these species, and thus modify their relationship with the environment, then isolation should be used for moderate or high risk.

Experiments leading to increased resistance of human pathogens to antibiotics or disinfectants could be performed only under moderate or high risk safety conditions, depending on the virulence of the organism involved.

Animal viruses: Experiments involving attachment of viral genomes or gene segments to prokaryotic vectors and their propagation in prokaryotic cells should be performed with host-vector systems, with a capacity of developmentzero outside laboratoryand with adequate safety measures for moderate risk. Rigorously characterised and purified segments of demonstrably non-transforming non-oncogenic viral genomes of oncogenic virus DNA can be attached to currently existing and propagated vectors within the enclosure required for moderate risk; where safer host-vector systems are available, such experiments can be carried out with low-risk measures.

Experiments aimed at introducing or propagating non-viral DNA or other low-risk agents into animal cells should use only low-risk animal DNA (e.g. viral, mitochondrial) as vectors and manipulations should be performed where moderate-risk isolation measures are in place.

3. Eukaryotes: The risks associated with the serendipitous attachment of eukaryotic DNA fragments to prokaryotic DNA vectors and the propagation of these recombinant DNAs in prokaryotic hosts are the most difficult to assess.

A priori, DNA from warm-blooded vertebrates is more likely to contain viral genomes, potentially pathogenic to humans, than DNA from other eukaryotes. Therefore, attempts to clone DNA segments from such animals and particularly primate genomes should only be made with host-vector systems that have a demonstrated capacity for restricted growth outside laboratoryand with isolation measures suitable for moderate risk. Until cloned segments of warm-blooded vertebrate DNA are fully characterised, they should be maintained in the most restrictive host-vector system in moderate-risk containment laboratories; when such cloned segments are characterised, they can be propagated, as suggested for purified segments of viral genomes.

Unless the organisms synthesise a known dangerous product (such as toxins, viruses), recombinant DNAs from cold-blooded vertebrates and all other lower eukaryotes can be constructed and propagated with the safest host-vector system available, with isolation measures for low risk.

Purified DNA from any source, which performs known functions and can be judged to be non-toxic, can be cloned with ordinarily available vectors and with low-risk safety measures. (The term toxic applies here to toxins, potentially oncogenic products or substances that could disrupt normal metabolism, if produced in an animal or plant by the presence of a micro-organism).

4. Experiments that should be deferred: There are feasible experiments that present such serious dangers that they should not be undertaken for the time being with the host-vector systems now available and with current protective measures. These include the cloning of recombinant DNA derived from organisms, which are considered to be highly pathogenic (i.e. etiological agents of classes III, IV and V according to the classification of the US Health, Educationand Welfare department), DNA containing toxic genes, and large-scale experiments (more than 10 litres of culture) using recombinant DNA capable of synthesising products potentially harmful to humans, animals or plants.

Realisation

In many countries, national organisations are beginning to take steps towards the development of codes or standards for the conduct of experiments with known or potential risks. Until these are fully established, we urge scientists to use what is proposed in this document as a guide. In addition, there are some recommendations that could be implemented immediately and directly by academic community.

developmentof vectors and safer hosts

An important and encouraging achievement of meetingwas the verification that special bacteria and vectors with restricted ability to multiply outside laboratorycan be genetically constructed, and that the use of these organisms will increase the safety of recombinant DNA experiments by many orders of magnitude.

Experiments in this direction are in progress, and bacteriophage variants, non-transmissible plasmids and special strains of Escherichia coli will be available in the near future. All these vectors could dramatically reduce the potential risk while helping to improve methodology. Other host-vector systems, especially modified strains of B. subtilis, bacteriophages and plasmids, may also be useful for specific purposes. It is quite possible that suitable and completely harmless vectors will be found for eukaryotic hosts, such as yeast, animal and plant cells that can be easily cultured. There is likely to be a continuing developmenton this areaand the participants of the meetingagreed that host-vector systems in which improvements are made that reduce the risks of recombinant DNA researchwill be available to all interested researchers.

Procedures for laboratory

laboratoryIt is the responsibility of the lead researcherto inform staffof the risks of such experiments before they are initiated. Free and open discussion is necessary so that each participant in the experiment fully understands the nature of the experiment and any risks involved. All staffshould be trained in safety measures to control risks, including emergency actions, in the event of an unexpected risk. It is also recommended that appropriate health checks, including serological monitoring, are carried out regularly for all staff.

exchangeexperience and training courses

The researchon this areawill advance rapidly and the methods used will find application in many different biological problems. It is impossible to foresee the full range of possible experiments and to make a precise judgement on each of them. Therefore, it is essential to carry out a continuous evaluationof the problems, in the light of new scientific knowledge. This could be achieved through a series of meetings of workand annual conferences, some of which should take place at international level. There should also be training courses for staffin the important methods, as this is likely to be of interest to subjectfrom worklaboratories that may not have had extensive experience in this area. Priority should be given to researchthat can improve and assess the effectiveness of isolation measures for existing and future host-vector systems.

New knowledge

This paper presents a first evaluationof the potential risks in the light of current knowledge. However, very little is known about the viability of bacterial strains and bacteriophages from laboratoryin different ecological niches in the outside world. Even less is known about whether recombinant DNA molecules will improve or worsen the survival of their vectors and hosts in nature. These questions are fundamental to the evaluationof any new organisms that may be built in the future. A researchneeds to be undertaken at this areaand given high priority. However, most molecular biologists who can construct recombinant DNA molecules are not doing these experiments and it will be necessary to facilitate a researchat partnershipbetween them and groups experienced in the study of bacterial infection or ecological microbiology.

A workshould also be developed to make it possible to monitor the escape or spread of cloned vehicles and their hosts.

Nothing is known about the potential infectivity in higher organisms of phages or bacteria containing eukaryotic DNA segments and very little is known about the infectivity of DNA molecules themselves. Transformation Geneticsof bacteria undoubtedly occurs in animals, suggesting that recombinant DNA molecules may retain their biological potency in this environment. There are many questions in this field whose answers are essential for a correct evaluationof the risks of experiments with recombinant DNA molecules. It will be necessary to ensure that this workis planned and carried out; and it will be especially important to have such information before large-scale application of the use of recombinant DNA molecules is attempted.