3.1 What is 'genetic modification'?

Genetic modification is officially defined as the alteration of genetic material (DNA or RNA) of an organism by means that could not occur naturally through mating and/or recombination [18].

3.2 EU Directives

Throughout most of the world the use of all live genetically-modified organisms (GMOs) is controlled by law. There are currently two relevant sets of regulations (Directives) governing genetic modification throughout the European Union. Laws in the United Kingdom have been enacted to comply with these Directives. Directive 98/81/EC covers 'Contained Use' e.g., work in a laboratory; the other (Directive 90/220/EEC) covers 'Deliberate Releases' of modified organisms into the environment e.g., field trials of genetically-modified crops. It is important to note that it is not the techniques of genetic modification that are controlled, but rather activities with living organisms produced by these procedures.

3.3 Microbial transformation

In the school context, work falling within the scope of these Directives is most likely to involve the 'transformation' of microorganisms, that is, the introduction of DNA into microorganisms by 'artificial' means. For pre-university educational work, this almost always involves the use of plasmid DNA. Plasmids are small rings of DNA comprising just a few genes, that are found in bacteria and yeasts. They are not normally essential for the microbes, but they may help them to survive in rare and exotic environments. For instance, some plasmids enable the bacteria that carry them to resist the toxic effects of heavy metals or antibiotics, or to live on particular nutrients. Sequences of DNA can be 'spliced' into plasmids, allowing them to be used as vectors for transferring genes between organisms.

Numerous practical kits have been developed for demonstrating microbial transformation, particularly in the USA where they have become a routine part of high school biology courses. A search of the World Wide Web will unearth many sites describing practical exercises for schools.

IMPORTANT
Although many of these procedures may be freely used in the USA, within the European Union such work is more strictly regulated, and teachers could easily be in breach of the law were they or their students to carry out the majority of the genetic modification exercises that are currently described on the Web.

3.4 UK regulations

In England, Wales and Scotland, genetic modification of organisms in containment is covered by the Genetically Modified Organisms (Contained Use) Regulations, 2000 [18]. Northern Ireland has its own separate but virtually identical legislation. Similarly, within Great Britain and Northern Ireland there are separate regulations covering deliberate releases of GMOs into the environment.

Before genetic modification (other than 'self-cloning', as defined below) is undertaken in the UK the premises involved must be registered with and approved by the HSE. There is a fee for this, and in addition, a local 'Genetic Modification Safety Committee' (GMSC) will need to be established, comprising individuals who are suitably qualified to advise on any risks to human health and the environment of all activities before they begin. Records of such assessments must be retained for at least 10 years after the relevant activity has ceased. Schools wishing to undertake such work are advised to contact the HSE for further details. These stringent requirements would seem to preclude most schools from carrying out practical genetic modification. There is currently one important exception to this rule, namely, 'self-cloning'.

3.5 'Self-cloning'

Microbial transformation in which DNA (or RNA) is returned to a species in which it could naturally occur is known technically (and rather confusingly) as 'self-cloning'. In this context 'cloning' means making copies of plasmid DNA within an organism. Because the plasmids used are made entirely from DNA that could occur naturally within the species involved, the work is called ''self-cloning'. The official definition of self-cloning runs as follows:

" ... the removal of nucleic acid sequences from a cell ... followed by the re-insertion of all or part of that nucleic acid ... into cells of the same species or into cells of phylogenetically closely-related species with which it can exchange genetic material by homologous recombination." [18]

In other words, if the transfer of genetic information is largely confined to that which could naturally occur within a single species, the work is regarded as 'self-cloning'. The nucleic acid may have been subject to modification by enzymatic, chemical or mechanical steps so as to produce a novel order of genes / bases, to remove sequences, to produce multiple gene copies, etc.

Self-cloning, where the resulting organism is unlikely to cause disease in humans, animals or plants, is exempt from the 'Contained Use' regulations. Schools and others may undertake such work without licensing their premises or setting up a GMSC. However, somewhat unusually (since these microbes could in theory be found in nature) the organisms produced are covered by the 'Deliberate Release' regulations.

3.6 Containment

Under current legislation it is an offence to release any GMO into the environment or to allow it to escape without prior consent of the Secretary of State. It is therefore essential that even 'self-cloned' organisms and adequately contained and that a 'release' does not occur. A key point is that an accidental release of a GMO might be considered to be deliberate if the steps taken to ensure containment are deemed to have been inadequate. Note that if a GMO cannot survive in, or transmit genes to other organisms in the environment, it is regarded as being 'biologically contained', and an accidental escape is not regarded as a 'release'.

Fortunately, containment can be ensured simply by following good microbiological practice and good occupational safety and hygiene, coupled with the careful selection of suitable host organisms and plasmids. This would usually involve, for example, using host strains that are weakened and 'non-mobilisable' plasmids that cannot transfer their genes into the host's chromosome, or be transferred into other organisms by natural means such as bacterial conjugation.

Kits from reputable suppliers, that have been designed for use in UK schools, should comply with these requirements.

3.7 Host strains

The species of bacterium that is most commonly-used for cloning work is Escherichia coli, strain K12. Unlike the wild type, K12 strains of E. coli are usually unable to inhabit the mammalian gut. This strain's origins can be traced back to work in the USA in 1922. Biochemical and genetic studies by Edward Tatum in the 1940s made the strain popular with researchers, and after many millions of generations of laboratory cultivation, it is now known to have undergone significant changes. These have altered the lipopolysaccharides that comprise the outer membrane of the bacterial cell, so that it can no longer infect mammals.

Many strains of E. coli K12 have been specially-selected for transformation work. Usually these do not harbour any extra-chromosomal DNA of their own, but can be transformed efficiently by plasmids. Compared to the wild type E. coli, these 'cloning strains' are severely weakened and would find it difficult to thrive outside the laboratory. They may have unusual nutritional requirements, and are often susceptible to damage e.g., from the ultraviolet component of sunlight.

3.8 Plasmids

Plasmids can pass from one bacterial cell to another of the same or a related species by a natural 'mating' process called conjugation. During conjugation, a tube or pilus is formed between adjacent cells, through which the plasmid passes. The genes required for the formation of the pilus are also carried on a plasmid (an F or fertility plasmid). Host strains used for transformation experiments in schools usually have no F plasmid, so that they cannot pass on genetic material by conjugation. They often also lack phages, so that DNA cannot be picked up and passed on by viral infection (transduction).

The use of non-conjugative strains of bacteria that lack phages, coupled with the use of non-mobilisable plasmids (see 'Missing genes', below) significantly reduces the risk of DNA being transferred between microorganisms, and hence the unwanted transfer of characteristics such as antibiotic resistance [20].

The transformation of bacterial cells with plasmid DNA is very inefficient, and only a small proportion of the cells treated will take up the DNA. Therefore a means of selecting those cells that have been transformed is needed. The incorporation of one or more antibiotic-resistance genes into the plasmid DNA used to transform cells is the commonest method of achieving this. In the presence of appropriate antibiotics, such plasmid-bearing cells thrive while their less well-endowed (untransformed) neighbours perish. In this way, selection pressure is applied to maintain the plasmid in the population of cells. Without that pressure, the few transformed cells would be swamped by their untransformed neighbours.

3.9 Missing genes

For a plasmid to travel through a pilus, two additional requirements must be met. The plasmid must possess a gene encoding a mobility protein (mob) and have a nic site. The mobility protein nicks the plasmid at the nic site, attaches to it there and conducts the plasmid through the pilus. Plasmids for school demonstration experiments usually have neither a nic site nor the mob gene. This means that once it has been introduced into a bacterial cell by artificial means (transformation) a plasmid cannot naturally transfer (by conjugation) into other cells that do not posses it.

3.10 Incubation at 37 °C

Although school texts sometimes warn against doing so, the delicate strains of E. coli used for cloning work often require incubation at 37 °C for speedy growth. Good microbiological practice, coupled with the use of selective growth media will ensure that contaminating human pathogens are not inadvertently cultivated at this temperature.

3.11 Physical and chemical containment

In addition to the biological containment measures described above, good microbiological practice must be followed to ensure that the microorganisms are physically contained during the investigation and destroyed afterwards. UK law requires that genetically-modified microorganisms must be inactivated after use by a validated means [21]. In practice in a school, this means that any cultures must be destroyed by autoclaving them. The containment and the destruction of cells when such work is undertaken will prevent the spread of antibiotic-resistant populations. In addition, most of the antibiotics used for such work are heat-labile and readily break down when media are autoclaved after use. Together, these methods of physical, chemical and biological containment will ensure that educational exercises demonstrating the principles of genetic modification are as safe as possible.

EXCEPTIONS TO THE RULE?

The definition of 'self-cloning' in the current regulations is slightly wider than that in the previous ones.

The combination of genes from different species may fall within the 'self-cloning' definition if the sequences concerned are markers "with an extended history of safe use in the particular organism concerned" [18].

The HSE judges each case on its own merits, and teachers are advised to consult with the suppliers of kits, etc. if they require clarification as to the legal status of a particular school laboratory exercise within the UK.

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