A beginner's guide to genetic engineering

(This text contains two large tables which are best viewed with the full width of the browser window.)

 

The following guide assumes no previous knowledge of the subject. For the meaning of words not explained in the text below, please see Ifgene's glossary.

 

What is genetics?

Genetics is the scientific study of genes, i.e. variations in the characteristics -- resemblances and differences -- of organisms and how these characteristics are inherited from generation to generation. Modern genetics is as much concerned with the organismic level of this process as it is with the cellular and molecular levels.

 

What are genes?

Whole articles have been written trying to answer this question. The conclusion is that a gene is a fuzzy concept which depends upon who is using the term and in what context. For the earliest geneticists, genes were fairly distinct traits or characteristics which could be observed in the whole organism. For the modern molecular biologist or molecular geneticist a more chemical definition of a gene is used which brings in a number of additional concepts that we explain below. The molecular gene is a definite sequence of bases in the DNA chain which together code for the production of a particular protein.

cell&dna.gif (27258 bytes)

The above diagram is an artist's symbolic impression using a false perspective of structures supposed to be present on a microscopic and molecular scale. The expert would notice that the diagram shows a great deal of artistic license. Nobody knows what these structures actually 'look' like in a living organism. Organisms are made up of single cells. The figure shows at the top a cross-section of a typical cell from an animal. At the centre of the cell is a nucleus which contains nearly all the genetic material. This genetic material is 'packaged' into organised structures called chromosomes, named after the way they take up coloured stain when prepared for examination by light microscopy at a particular stage of the cell's cycle of growth and reproduction. The diagram shows a chain of DNA (desoxyribosenucleic acid) unravelling out of one of the chromosomes. Note its double helical geometric structure shown as two intertwining ribbons. Lying between the two backbones of the double-helix are chemical substances known as bases: guanine (G), thymine (T), cytosine (C) and adenine (A). Through the chemical nature of the bases they pair up with each other in the following specific way: G with C, and T with A. The title of the film 'GATTACA' is inspired by the initials of these bases. The base pairing is a reversible linkage which is strong enough to hold the two coils of the double helix together in a definite geometrical arrangement. Genes are made of definite sequences of bases on the DNA chain which code for the production of particular proteins. Proteins are chemical substances which mediate the form and function of cells and organisms either by forming part of definite structures or by acting as biological catalysts in living processes. More on genes, DNA and proteins etc can be found at the following sources:

Text boxes in article on Ifgene web site http://www.ifgene.org/heaf3.htm  

DNA From The Beginning -- an on-line primer provided by Cold Spring Harbour Laboratory

Access Excellence -- Genentech's molecular biology education pages

 

What is genetic engineering?

Engineering is the technological manipulation of the objects of the natural world in a way that is perceived to be beneficial to people. Traditionally we used the word in the context of inanimate nature: bridges, railways and machines etc. But the term can be used and is used in the context of biology, namely for bioengineering, i.e. modifying or manipulating living organisms. Another term used in place of the term 'genetic engineering' is 'biotechnology'. Some people think that 'biotechnology' sounds less emotive, less fearful. How is genetic engineering defined then? As with the term 'gene', it depends upon who is using it and in what context. The following table illustrates how difficult it is to answer this question in a simple sentence. The table includes most forms of artificial interference in the outcome of reproductive processes. (Link to Ifgene glossary -- a list of the meanings of words)

Summary of techniques affecting the outcome of breeding/reproductive processes

Microorganisms

Plants

Animals

Man

Manipulation of bacterial conjugation, e.g. interruption by a mechanical process. (Not regarded as genetic modification in EU regulations) Selection/gathering of species or varieties of wild species dating back to the beginnings of farming some 10,000 years ago

Deliberate encouragement of specific crosses, e.g. Alexander the Great's eugenic intermarriage of Greeks and Persians. See also Plato's Republic, Book V 459e et  seq.

Bacterial transduction, i.e. use of phages (bacterial viruses) to carry bacterial or other genes or artificial DNA sequences into bacteria. (Not regarded as genetic modification in EU regulations) Traditional breeding: deliberate crossing of varieties of species followed by selection with or without specific environmental challenges, e.g. cold, salty soils, diseases. Traditional breeding: deliberate crossing of varieties of species followed by selection -- i.e. prevention of undesirable crosses. Selection of newborn through environmental challenges, e.g. (as legend has it) in ancient Sparta. Selection by infanticide.
Transformation, the natural tendency of bacteria to take up DNA in their environment, can be used to insert DNA functional as genes into the bacterial genome resulting in transgenic bacteria, e.g. bacteria transformed so as to make 'human' insulin for treatment of diabetes. (Bacterial transformation is not regarded as genetic modification in the EU regulations) Crosses which cross species barriers, often necessitating 'embryo rescue' to construct a viable plant (e.g. Triticale, a wheat-rye hybrid). Crosses which cross species barriers, e.g. mules & hinnies. Genocide (e.g. Adolf Hitler's 'final solution')
Artificial control of pollination, pollen transfer, prevention of self-pollination, covering up selected flowers to restrict pollination.

Artificial insemination with careful selection of the donor animal.

Artificial insemination, the woman sometimes selecting sperm stored in a sperm bank from a donor with known characteristics (e.g. high IQ).
Male sterilisation by anther removal or insertion of male sterile genes. Sterilisation (e.g. castration) Sterilisation. Compulsory sterilisation of selected individuals is still sanctioned legally in some countries.
Polyploidy induction: i.e. chromosome doubling, tripling etc with e.g. colchicine. Surrogate motherhood
Creation of specific hybrids to exploit the heterosis effect, i.e. hybrid vigour. F1 hybrids usually do not breed true or are sterile. Manipulation of animal embryos in vitro to mix the cells and create chimaeras, e.g. the geep, a mixture of sheep and goat.  (See 'History.htm' for an image) Amniocentesis, chorionic villus sampling or sampling foetal cells in umbilicus wall or maternal blood, followed by genetic or cytological testing, followed by abortion of undesirable embryos, foetuses.

Chemical or radioactive mutagenesis , i.e. creation of mutations (changes) in the genes artificially.

Contraception. Abortion after genetic testing (e.g. for Downs syndrome, trisomy 21).
  Anther culture in vitro. Many methods of plant reproduction involve a cloning or vegetative propagation step on cells or the whole organism (e.g. growing potatoes). Nuclear transfer 'cloning' (e.g. Dolly the sheep [See 'History.htm' for an image] and many other species). Nuclear transfer 'cloning' (illegal in many countries) for various purposes (e.g. stem cell therapy). Cloning, i.e. induction of twinning etc by physical manipulation of embryo prior to implantation.
Somatic cell culture, protoplast (plant cell wall chemically removed) fusion.
In-vitro fertilisation and selection (not regarded as genetic manipulation in EU regulations). In vitro fertilisation (IVF) usually after induction of super-ovulation in the animal. In vitro fertilisation (IVF, 'test tube' babies).
Marker assisted selection, i.e. a genetic marker meaning a particular DNA sequence is used to select organisms for further breeding. It avoids having to wait for the organism to reach maturity to see expression of the characteristic linked to the marker. IVF followed by genetic testing of one cell of the developing embryo followed by implantation in mother or surrogate only if genetically desirable (Pre-implantation genetic diagnosis, e.g. Adam Nash who as an embryo was found to be free of the hereditary disorder Fanconi's anaemia and whose umbilical cells were used after he was born to cure his sister Molly of the disease).
Creation of transgenic plants (background article),  e.g. by switching off genes (Flavr Savr tomato); insertion of genes from unrelated species (e.g. Bacillus thuringiensis insect toxin gene) or even from animals; inclusion of control of expression genes; insertion of selectable marker genes (e.g. genes for antibiotic resistance). Creation of transgenic animals (e.g. Herman the bull, Tracy the sheep [See 'History.htm' for images]) for pharmaceutical production, xenotransplantation (transplantation of animal tissues/organs into humans) or  research into diseases (e.g. mice with certain genes 'knocked out'). Intracytoplasmic sperm or spermatid (immature sperm cell) injection (ICSI).
Gene therapy, i.e. insertion into embryo, child or adult of specific genes (DNA sequences) using a suitable vector (carrier). Potential for so-called 'designer babies'. This includes 'germ line' gene therapy where the descendents of the patient would be affected.

Whilst the term 'genetic engineering' is generally used for recombinant DNA technology or transgenesis, i.e. artificially 'cutting' DNA and 'splicing' it into an organism's DNA (the bottom row of the table), the above table caters for those biotechnologists who love to claim that people have been doing genetic engineering for thousands of years. In the remainder of this guide we shall concentrate on the recombinant DNA or transgenic aspect of genetic engineering, as it is this which is causing most concern. Organisms formed in this way are usually referred to as 'genetically modified organisms' (GMOs). Other terms which are synonymous with 'genetic engineering' are 'gene technology' and 'genetic technology'.

 

How is genetic engineering done?

First catch your gene

For a detailed answer to this question the reader is referred to the various educational sites linked from our 'other web sites' page. Here we concentrate on a general introduction. The DNA that one wants to recombine with other DNA in the organism of interest has first to be extracted from the cells of the organism. It does not have to be from the same species. 'Human' genes could for instance be inserted into plants. The extraction is done by physical and chemical methods. Alternatively, if the DNA sequence of the gene of interest is known, it can be made from the single units which hold the bases A, C, G or T.

If there is insufficient extracted DNA for further manipulation it can be exactly copied and thus reproduced in the test tube exploiting the property of bases in the two complementary strands of the DNA helix to pair with each other in a precise way. The procedure used for doing this is the polymerase chain reaction (PCR) named after the polymerase enzyme which catalyses the building up of new DNA chains from its building blocks, the single units which hold the bases. It is precisely this method which is used in DNA-fingerprinting, a technique used by forensic sciences to identify individuals who were at the scene of a crime and may have committed it.

Finding the gene or DNA-sequence of interest in an organism's DNA is a bit like trying to find a needle in a haystack. The DNA has to be got into some form where it can be 'filed', catalogued and retrieved at will in sufficient quantities for further work. A convenient way of doing this is to clone the DNA in microorganisms such as yeast or bacteria, thus creating living 'libraries' of DNA. The procedures exploit the normal reproductive properties of the microorganisms, such as transformation and transduction in bacteria. Migroorganisms can be manipulated in a precisely controlled way in the laboratory and have the advantage that their generation or doubling time is short, e.g. about 20 minutes for bacteria. Unused organisms can also be stored frozen indefinitely in a viable form for immediate use. This part of the genetic engineering process is carried out in laboratory facilities which should provide complete containment of the organisms and such procedures are controlled by the 'contained use' GMO regulations. Different stringencies of contained use apply according to the level of perceived danger from the GMO.

To find the particular gene of interest the gene library has to be searched with a suitable probe. Just like finding a book in a library one needs to have a bit of an idea about what one is looking for, so too, in searching a gene library one needs to know something of the nature of the gene one is looking for. Such information can come from classical genetics, for instance the gene could be closely linked in the process of inheritance to another which has already been identified. Or one can construct a probe by working backwards from the protein, the known expression product of the gene. By knowing the sequence of amino acids, the building blocks of proteins, in the protein or in part of the protein it is possible to work out from the genetic code for each amino acid, i.e. the sequence of three bases which codes for it, what the sequence of DNA is in the gene that codes for the protein. A short DNA probe can then be constructed which has the property that it will bind relatively strongly to the complementary strand of the DNA of the gene of interes in the gene library. Various blotting, washing, transfer and chemical methods are used with the organism in the gene library and the DNA obtained from it to find the gene of interest. In practice a whole collection of methods are used gradually to home in on the gene. It must be obtained intact, that is in a form where it could given the right circumstances produce its normal protein expression product. To make sufficient quantities of it for further work it can be multiplied by PCR (see above).

Getting the gene into the organism

We have already looked at some ways to get genes into microorganisms. Here we look at methods for plants, animals and human beings. In many applications a microorganism is used as a carrier or vector to 'smuggle' the gene of interest into the target organism. A common method for getting genes into plants is given in one of the articles on this web site from with the following is quoted with minor editing:

"How to make a genetically modified  plant is illustrated in the diagram below using a simplified textbook example involving a bacterium as the gene carrier.

Heaf.gif (20450 bytes)

But the transgene does not always have to be smuggled in by an infective organism. Ballistics, i.e. using a so-called 'gene gun', allows the DNA containing the gene, coated on microscopic gold or tungsten particles, to be blasted into plant cells. Once inside the cell nucleus, natural processes allow incorporation of the foreign DNA into the plant's own DNA in its chromosomes. If the inserted gene has the right control genes attached it will trigger the production of the particular protein that corresponds to it, which in our example is a bacterial enzyme that replaces the plant's own enzyme whose action is stopped by the herbicide. [...]

So far, the most popular transgene apart from antibiotic resistance marker (ARM) genes has been for herbicide resistance which have now been put in soybeans, maize, wheat, rape, fodder/sugar beet and chicory. It allows the crop to be sprayed with a total herbicide only if weeds are becoming a problem without killing the crop. The second commonest transgene is for insect resistance. The soil bacterium Bacillus thuringiensis (Bt) produces some 150 insect toxins, a cocktail of which is widely used in organic agriculture outside the UK to spray crops to kill insect pests. The genes coding for the toxic proteins have been isolated and transferred to crop plants such as maize, potato and cotton. If insects eat the leaves they are killed. The most widely advertised transgene is one which prevents the expression of a softening enzyme in tomatoes allowing them to be ripened on the plant whilst reaching the supermarket sufficiently firm for sale. Some of the hundreds of other transgenes tried or in the pipeline include leaf-roll virus resistance, insect resistance from snowdrop and modified starch content in potatoes; altered lignin content in the poplar tree; modified oil content, reduced pod shatter, fungal tolerance and male sterility in rape; insect resistance (Bt toxin) in tomatoes; and Arctic turbot antifreeze protein genes in strawberries." (Taken from www.ifgene.org/heaf.htm)

Genes can be injected directly into the nucleus of animal cells (pronuclear injection). Usually a gene construct is used which comprises the gene of interest and any necessary additional pieces of DNA which provide the insertion and control/regulatory sequences. Control sequences are necessary to trigger the expression of the gene of interest, i.e. the making of the protein which corresponds to it. In the case of sheep producing alpha-1 antitrypsin (an enzyme needed for normal lung function) in their milk for extraction and use as a pharmaceutical in the treatment of emphysema, the control sequence has to be one for a normal milk protein in sheep. The insertion of genes into animal cells can be conveniently carried out at the stage of the embryo during normal IVF procedures.

However, this is not yet permitted for human cells. It would amount to germ line genetic engineering, i.e. the descendents of the modified individual could end up having the inserted gene plus any complications caused by the insertion process. However, there has already been much research and many clinical protocols for the genetic modification of parts of human beings for the purposes of therapy, i.e. gene therapy. By far the most important current application for gene therapy research is in the treatment of cancer. The aim here is to use gene constructs specifically to target cancer cells, i.e. cells which show unregulated growth, and either kill them or bring them under the body's normal control. One type of gene therapy of immediate interest is in the treatment of cystic fibrosis. Sufferers are unable to make a protein important to normal function of lining of the lung and other connective tissues in the body. Treatments have been focused on the lung where the severest and most life-threatening symptom lies. One way of administering the gene is as a nasal spray with the gene incorporated into an adenovirus, the common cold virus which infects the respiratory tract lining and inserts itself into cells. The virus is attenuated to reduce its disease causing tendency, but this nevertheless remains a risky method of treatment. Another, less dangerous, approach is to encapsulate the gene in liposomes, minute globules of fatty substances with a specially designed structure. Under certain conditions these are engulfed by cells, digested and the DNA in them released into parts of the cell where it can be incorporated into the rest of the cell's DNA. Links to gene therapy sites can be found on our 'other web sites' page.

Fate of inserted genes

Genetic engineering is often described by its proponents as a very precise method of changing the genetic makeup of an organism. Under ideal conditions a precisely defined number of copies of a pure gene attached to only those control sequences which are absolutely necessary would be inserted at a known locus or place in the organism's genome (the totality of its genes/DNA) in a way which is stable and does not disturb the organism's integrity. The gene would then produce its expression product protein which would take its place in a way that harmonises with the form and function of the cell and whole organism. In actual practice, this ideal is rarely achieved. For many GMOs the fate of the inserted genes in the organism can only be unravelled by painstaking analysis. A variable number of copies ends up randomly peppered throughout the host genome. In the gene uptake process, parts of the gene construct can get lost. In cases where this happens to an antibiotic resistance marker gene attached to the gene of interest, the loss can be an advantage. Nobody wants antibiotic resistance genes spread further than their natural limits. Furthermore, inserted genes can be immediately or gradually 'silenced' by normal DNA management mechanisms of the host organism. This little understood silencing process basically switches off the gene thus preventing it from expressing and usually occurs by chemical alteration. As the knowledge of molecular biology advances it is likely that insertion of genes will be more precise and happen in such a way that gene silencing is avoided. Gene targeting is a more precise way of inserting genes at known loci in the genome. The technique has been used successfully to create transgenic sheep.

Another concern about inserted genes is the extent to which they disrupt the integrity of the host genome or interfere with the normal expression of the host's genes. However, any GMO intended for commercial use goes through an extensive series of tests which more or less thoroughly establish whether the organism is 'substantially equivalent' to the parent organism. But the application of this term is highly controversial. Critics argue that GMOs are not at all equivalent to the parent species. One example would be a protein product of a transgene expressed in a food plant, for instance a herbicide tolerance or insect resistance gene. As such proteins may be foreign to a normal diet they may cause allergies.

 

What are the issues in connection with genetic engineering?

A detailed list of the pros and cons of genetic engineering is provided on this web site. Here we offer only bullet point abbreviated lists as a guide to further reading. Many of the purported benefits and dangers are as yet highly speculative. The technology is still in its early days. We retain in the following table the divisions used above, namely microbial, plant, animal and human applications.

Benefits and disadvantages/dangers of genetic engineering (Link to Ifgene glossary)
Microorganisms Plants (Food) Animals Man
Disadvantages/dangers
  • horizontal gene transfer
  • creation of dangerous novel pathogens and diseases which may escape containment
  • spread of antibiotic resistance
  • viral recombination of supposedly crippled viruses
  • creation of new invasive species (super-weeds)
  • contamination of organic/non-GM crops/food with transgenes
  • loss of biodiversity of farm wildlife
  • loss of crop genetic diversity increases famine risk
  • more dependence on herbicides
  • increase in herbicide use to control volunteers (weedy crop plants)
  • horizontal gene transfer into soil and gut microorganisms
  • spread of antibiotic resistance marker genes
  • genomic instability
  • allergenicity of foods
  • novel toxicity of foods
  • crossing 'species barriers'
  • chemical/seed corporations control food supply
  • toxins from GM plant debris affect soil flora/fauna
  • genetic instability of transgenic lines
  • increased risk of pesticide/herbicide resistance
  • live recombinant (cancer) viruses from virus-resistant GM plants
  • genetic pollution multiplies
  • unsustainable agriculture
  • violation of integrity of animal (crossing species barriers)
  • increased instrumentalisation of animals
  • animal suffering
  • zoonoses (GM animal to human disease transfer in xenotransplantation)
  • creation of new viral diseases through transfer
  • a new tool for eugenicists (IVF, diagnosis, selection)
  • iatrogenic death/illness from gene therapy
  • potential for creating a genetic underclass
  • instrumentalisation of the one individual by others ('designer babies')
  • transmission of adverse effects of germ line manipulation
  • creation of GM human beings (embryos) to supply body parts
  • iatrogenic illness from drugs made by genetic engineering, e.g. insulin
  • risk of mutation from trans-species transfer of 'jumping genes'
  • creation of superviruses by viral recombination in human cell culture
  • cancer from gene therapy viral vectors recombining with endogenous viruses 

Benefits

  • versatile tool for gene manipulation in preparation for making other GMOs
  • creation of new pharmaceuticals which cannot be made in any other way
  • ease of containment
  • bioremediation (cleaning up waste/pollution with living organisms)
  • quicker, more predictable way to generate new cultivars
  • increased biodiversity of weed populations and hence farm wildlife
  • overall herbicide and pesticide use lower
  • cultivar properties better known than ever before
  • genes from different genera can be inserted
  • helps food security
  • gene flow (pollution) controllable (male sterility, terminator genes)
  • sustainable agriculture
  • reduced tillage and fuel consumption
  • production of vaccines and other drugs in plants
  • phytoremediation
  • GM animals can generate pharmaceutical proteins which cannot be made in any other way
  • new agriculturally useful GM breeds, e.g. which can tolerate factory farming without suffering
  • treating genetic disorders and cancer
  • creating new types of human beings with advantageous traits
  • supplying body parts

Genetic engineering in its modern transgenesis form has been around for only 25 years. From its beginnings in the 1970s it has been hotly debated. The above table shows only some of the key claims and counter claims. In addition to these are many social consequences making the overall risk/benefit assessment highly complicated. Whilst so many perceived benefits and disadvantages await substantiation by further research or deployment of GMOs in everyday life, it is difficult to draw up a balance sheet. However, it is becoming increasingly clear that consumers of the products of the technology will settle many issues purely by the force of their purchasing power and freedom to choose. However, for that process to happen in a fully aware fashion, organisations such as Ifgene will have to continue their work of facilitating 'judgement forming and public awareness' about the subject of genetic engineering. One area which deserves more attention than it has received so far is the underlying scientific thinking, mind set, world view or ideology that makes genetic engineering possible in the first place. Coupled with this is an examination of the philosophical basis of the subject and this leads naturally to the realm of ethics and forming moral judgements. Some key topics for a deeper approach are:

For those who want to venture further, many of the articles on Ifgene web site examine these deeper aspects.  

 

Compiled by Dr David Heaf, Ifgene UK, 15th November 2000, slightly edited 23 April 2005
The URL of this page is http://www.ifgene.org/beginner.htm  

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