3.1. Genetic engineering

Unit 4: The genetic revolution 1 2 3 4

3. BIOTECNOLOGY

For thousands of years, mankind has domesticated animals, has improved crops and has used microorganisms to obtain useful products, such as bread, wine, cheese and yogurt. Biotechnology is not new, but until modern times it has been used in an empirical way and without scientific knowledge.

The modern biotechnology implies the deliberated manipulation of the genetic material (DNA) of the organisms with a practical aim.

The progress of the modern biotechnology is based on:

- the knowledge of the mechanisms that regulate the expression of the information stored in genes.

- the development of tools and techniques that allows the manipulation of this information.

The main biotechnological techniques are:

The genetic engineering, which allows isolating, sequencing and transferring genes between organisms.

The cellular cloning, which allows the reparation of damaged or defective tissues and organs.

The cultivation of cells and tissues, which allows growing in vitro cells, organs and embryos.

In next years, biotechnology will produce a wide variety of products which practical applications will be focus on human health, agriculture, livestock breeding, industry and environment.

3.1. Genetic engineering

The genetic engineering or recombinant DNA technology involves a series of techniques that allow:

To manipulate DNA (cut, glue, reproduce and sequence DNA fragments of an organism).

To insert a DNA fragment from a donor organism in other DNA molecule of a receptor organism. As a result a hybrid molecule of recombinant DNA is obtained.

3.1.1. The genetic engineering tools

In living cells, the DNA is cut and glue once and other by specific enzymes:

The restriction enzymes that cut the DNA. Each one recognises specific sequences of nucleotides.When they cut DNA they generate “sticky ends”

The DNA ligases that join segments of DNA, gluing their sticky ends.

In addition is necessary a vehicle which transport this recombinant DNA to the interior of a receptor organism. This transporter is known as transfer vector and can be a plasmid (small circular fragment of extrachromosomal DNA present in some bacteria) or a virus.

Animation: Restriction enzymes (McGraw Hill)

3.1.2. The genetic engineering techniques

a) DNA cloning

The cloning of a DNA fragment consists of the obtaining of millions of copies of it. The method is the following:

1. The plasmid and the DNA that we wish to clone are treated with the same restriction enzyme. Then they are joined to obtain a recombinant plasmid. This plasmid is added to a bacterial cultivation (usually Escherichia coli), in appropriate conditions to allow bacteria incorporate the plasmid. This process is named transformation.

2. Recombinant plasmids have a gene that gives them resistance to an antibiotic. So that the transformed bacteria can be selected putting them in a culture test plate with a medium with this antibiotic. Only bacteria with the recombinant plasmid will survive and will form colonies. The rest that do not contain the recombinant plasmid will die.

3. Each colony of bacteria is isolated and maintained in growth conditions. Each time bacteria divide, the recombinant plasmid and the inserted gene also duplicate.

Animation: Steps in cloning a gene (McGraw Hill)

Animation: Early genetic engineering experiments (McGraw Hill)

b) Analyses of DNA fragments

Once restriction enzymes have cut a DNA molecule, the result is a group of DNA fragments of different sizes. These fragments can be separated and analysed by means of different techniques. Agarose gel electrophoresis is the most effective one.

This technique separates the DNA fragments by size and electric charge. The substrate used is the agarose gel, a polysaccharide that transforms into jelly-like substance when is dissolved in boiling water and then it is cooled down.

The procedure is the following:

Firstly a thin layer of agarose is prepared in a mould that has a row of small wells.

Then the layer of agarose is submerged in an electrophoresis tray with a salts and water solution (buffer) that has electrodes at both extremes.

Inside each well a sample of fragmented DNA is deposited.

As DNA has negative electric charge, when electric current is applied, the DNA fragments displace through the gel towards the positive electrode, located in the opposite extreme of the tray.

Agarose acts as a sieve. The smaller fragments of DNA cross the gel faster and easier than those bigger. And after a time, the DNA pieces are separated according their size and length in decreasing order. Each formed band correspond to all the fragments of similar size.

This technique allows obtaining a striped pattern that is typical and exclusive of the DNA of any organism. This pattern is known as genetic fingerprint or genetic profiling and makes possible determine the identity of an individual by comparing its sample with other ones.

Its main use is in forensic medicine to the identification of criminals or carcasses, and determination of paternity or kinship.

Animation: Gel electrophoresis (Summanas)

Animation: Paternity test (Summanas)

Animation: How to obtain a DNA fingerprint (NOVA)

Virtual Lab: DNA extraction

c) DNA hybridisation (DNA probes)

The hybridization is a natural and spontaneous process by which two single-chained strands of DNA with a complementary sequence of nucleotides join to form a double-chained DNA molecule.

This property can be used in laboratory to identify the presence of a determine fragment of DNA or gene in a sample.

A DNA probe is an artificial fragment of single-chained DNA traced with radioactivity or fluorescence and which nucleotides sequence is complementary of the sequence of the gene we want find. By means of X-rays is possible detect the DNA prove hybridised.

When we want to analyse simultaneously thousands of genes, the technique used is the DNA microarray, DNA chips or biochips.

A biochip is glass layer divided into thousands of microscopic cells. A little amount of single-chained DNA fragments that act as DNA probes for a determine gene, is located in each cell. As we know the accurate situation of each DNA prove in the biochip, any fragment of DNA which hybridises with one of them could be easily identify by its position in the biochip.

The biochip technology is used to:

Detect mutations which can provoke illnesses (E.g. haemophilia, cystic fibrosis, etc.)

Control the gene expression in carcinogenic cells.

Diagnose infectious diseases (by identifying the pathogen)

Personalise the medical treatment (to avoid adverse reactions)

Animation: Microarrays (McGraw Hill)

Video: Using a DNA microarray

Animation: DNA chip

Virtual Lab: DNA microarray

d) DNA amplification or PCR

The PCR (polymerase chain reaction) is a chain reaction which creates millions of copies of a DNA segment through the repetition of multiple cycles of DNA replication in vitro.

The PCR is a technique used to increase the number of segments of DNA of a sample of blood, semen, or tissues obtained in a crime scene or in an accident, or the DNA of pathogen microorganisms.

Animation: PCR (McGraw Hill)

Virtual Lab: PCR

e) DNA sequencing

The determination of the sequence of nucleotides of a fragment of DNA is an essential part of the genetic engineering. The first methods to sequence DNA were complex and required long time.

Nowadays, these techniques have improved and are automatic and computerised. They allow a simple and quick obtaining of the type and order of the nucleotides of a DNA sample.

In this way, it has been possible to obtain the sequence of thousands of genes and complete genomes of numerous organisms, from prokaryotes to humans.

Animation: DNA sequencing (NOVA)

3.1.3. Applications of genetic engineering

a) Genetically modified organisms (GMO)

The genetically modified organisms (GMO) or transgenic organisms are organisms (bacteria, fungi, animals or plants) that contain one or more genes from other species. These genes are called transgenes. When the transgene express, it elaborates in the transgenic organism, the same protein that it elaborates in the original organism.

The use of “designed organisms” patented, both prokaryotes and eukaryotes, is more often each time. The goal is the production of useful proteins and the obtaining of plant and animal varieties with new interesting feature for humans.

1. Genetically modified microorganisms.

Environmental preservation.

- Bioremediation:This is the use of transgenic microorganisms to eliminate environmental pollution. Bacteria are used to:

- Eliminate oil spills through the use of bacteria able to digest petroleum hydrocarbons transforming them into harmless substances.

- Eliminate heavy metals from soil.

- Biodegradation of plastics.

- Biofuels production. It is possible use diverse microorganisms, such as yeasts to produce biodiesel and bio-alcohol, sources of energy less pollutant then fossil fuels.

Products manufacture (industrial, pharmaceutical and medical)

Genetically modified microorganisms are used as “living factories” too. They can be transformed to produce useful substances that they do not produce naturally. Some examples of these substances are:

- Enzymes are proteins that catalyse different biochemical reactions, among them the degradation of substances. For example detergents contain enzymes produced by bacteria and fungi genetically modified. These enzymes dissolve the spots of clothes.

- Antibiotics are substances naturally produced by some fungi, used to kill or inhibit the growth of bacteria. Today many antibiotics, such as tetracycline or penicillin are produced by fungi and bacteria genetically modified.

- Some human proteins used in medicine, such as:

- Hormones (insulin and growth hormone)

- Blood coagulation factors (haemophilia treatment)

- Antibodies or immunoglobulins (monoclonal antibodies)

- Interferon (antiviral)

2. Transgenic animals

Transgenic animals have in their cells a gene from other species. The transgene, that has been obtained by means of the DNA recombinant technique, is inserted in the receptor animal through different mechanisms, for example by microinjection in a fertilise egg.

Applications of transgenic animals are several, for example:

To increase the resistance to diseases and to improve the animal production. Genetic manipulation has got cows that grow and mature faster, sheep that produce finer wool, pigs that produce meat with less fat, or salmons that reach the double of their original size, for instance.

To design knockout animals. In these animals, a normal gene (functional) is substituted by another mutant one (non-functional) to determine the effects over the organism and to find out the function of this gen. Mice are the most used animals in this kind experiments because most part of their genes act in a very similar way to those of humans. Their manipulation can help knowing the functioning of genes responsible for cancer and so understand the evolution of this illness.

To make organs for transplants. The problem of the lack of human organs for transplants could be solved by making transplants from other species (xenotransplants). The most suitable species is pig, because its organs are similar in size to human ones and they are easy to breed. However, it is not able to make these transplants directly from a pig to a human because the human organism would not recognize it and it would be rejected. Genetic engineers are working in a program to obtain transgenic pigs in which the immune system is modified in order to human immune system recognize it as own. In this way organ rejection would be avoid in case of transplant.

To create pharmaceutical farms. The transgenic animals breed in these farms has been designed to produce a wide variety of pharmacological products or biological molecules used in medicine that would be much more difficult to obtain in other way. It is possible to insert a transgene that codify a specific human protein (insulin, coagulation factors, etc.) in the DNA of sheep, cows or goats. The product obtained is secreted with the milk produced by these animals.

3. Transgenic plants.

The features of the transgenic plants are very diverse:

Resistance against herbicides and plagues. There are plants with bacterial genes that have them resistance against herbicides (e.g. soya, corn, cotton, etc.) which allow them grow while weeds are eliminated by fumigation. Others have transgenes that synthesise toxic substances against insects which parasite them in natural way. This makes possible reduce the use of phytosanitary products (pollutant and harmful for health) and reduce the economics costs of the production.

Resistance against frosts, drought and acidity or salinity of soil. In this way is possible to avoid the losses because of climatic causes and the exploitation of non-suitable soils for agriculture.

Dilation in maturing. Fruits and vegetables can be collected, transported and stored with guarantee that they will arrive fresh to the consumer.

Improvement of the nutritional value of agricultural species. It has been designed plants that produce vitamins. This ensures the supply to population.

Production of pharmacological substances.Pharmaceutical plants produce vaccines and human proteins.

Animation: Transgenic plants

b) Diagnosis and prevention of genetic diseases

The genetic disorders are produced when the genetic material is altered (mutations) and it does not have a correct functioning. These alterations can transmit to the offspring when they are located in the gametes productive cells.

Genetic inheritance diseases can be:

Chromosomal diseases which affect the number or structure of the chromosomes. For example, Down syndrome is an alteration of the number of chromosomes in 21 pair. There are 3 chromosomes instead of two in this pair.

Genetic diseases when they affect just a gene. For example the cystic fibrosis is due to a mutation in a gene of the chromosome number 7.

The prevention of these diseases can be:

Primary prevention

It is made before the conception.

It is carried out through the genetic advice. The individual or couple that wishes to have a child and have some risk to transmit genetic disease, receive information about the possibilities that this happed and about the options they have to minimize them. .

Secondary prevention

It is made after the fertilisation.

It is carried out through the early diagnosis. This can be:

- Pre-implantation diagnosis. In this case, the genetic material of the obtained embryos by in vitro fertilisation is analysed before its implantation in the uterus. In this way is possible to select the healthy embryos.

- Prenatal diagnosis. This has the inconvenient to increase a little the spontaneous abortion rate (1%). It can be performed by two different techniques:

- Amniocentesis: Cells of the amniotic sac (with the same chromosomal dotation than the embryo) are extracted and then are cultured in laboratory. Later, its genetic material is analysed to find out possible alterations.

- Corionic villus sampling: Placental cells (with the same chromosomal dotation tan the embryo) are extracted and then analysed.

c) Gene therapy

The gene therapy has as aim to treat, cure and prevent diseases provoked by a single dysfunctional gene by introducing in the patient a therapeutic functional gene.

The insertion of the functional gene pretends replace the defective gene and repair the genetic anomaly, or proportion a new function to the cells that compensates the defects shown by them.

Somatic gene therapy.

With this technique it is tried to correct a disease by treating only some cells of the illness person. In this way, the existence of only a little number of transgenic cells can be enough to diminish the symptoms of the disease. Vectors, usually viruses are used to introduce the therapeutic gene in the target cells. The transference can be in vivo or ex vivo.

In in vivo technique, the recombinant DNA is introduced by a liposome or a virus that is administrated by aerosol or injection

In ex vivo technique cells are extracted and culture in laboratory with the recombinant DNA. The modified cells are later reintroduced.

Video: Gene therapy

Germinal gene therapy.

It consists of introducing new genes, biologically functional, in germinal cells (ova and spermatozoa) before the fertilisation.

The embryo starts to form from a zygote genetically modified, so that all the cells of the new individual, including the germinal cells that it will produce in the future, will be genetically modified and it could transmit these characteristics to its offspring.

Although this type of genetic engineering is performed in laboratory mice, at this moment it does not applied to humans due to the profound ethical implications it has.