Insulin: a point in favour for transgenics

Despite the arguments and positions against transgenics, it is undeniable that insulin is a great transgenic success. It is essential in some types of diabetes; and since it was discovered, the life expectancy of diabetics has increased more than 45 years. Therefore, let’s know in detail.

REMINDER OF GENETIC ENGINEERING

Genetic engineering allows to clone, that is, to multiply DNA fragments and produce the proteins for which these genes encode in organisms different from the one of origin. That is, if in an organism there is an alteration or mutation of a gene that prevents the genetic code from translating it into proteins, with the techniques of recombinant DNA a gene is obtained without the mutation in another organism. Thus, it is possible to obtain proteins of interest in organisms different from the original from which the gene was extracted, improve crops and animals, produce drugs and obtain proteins that use different industries in their manufacturing processes. In other words, through genetic engineering, the famous transgenics are obtained.

They offer many possibilities in the industrial use of microorganisms with applications ranging from the recombinant production of therapeutic drugs and vaccines to food and agricultural products. But, in addition, they have a promising role in medicine and in the cure of diseases.

And is that the result of obtaining a recombinant DNA, from it, will be made a new protein, called recombinant protein. An example of this is the case of insulin.

WHAT IS INSULIN?

Insulin is a hormone produced in the pancreas and with an important role in the metabolic process. Insulin comes from the Latin insulae, which means island. Its name is due to the fact that inside the pancreas, insulin is produced in the islets of Langerhans. The pancreas is related to the general functioning of the organism. It is located in the abdomen and is surrounded by organs such as the liver, spleen, stomach, small intestine and gallbladder.

Thanks to it we use the energy of the food that enters our body. And this happens because it allows glucose to enter our body. This is how it provides us with the necessary energy for the activities we must perform, from breathing to running (Video 1).

Video 1. Insulin, Glucose and You (Source: YouTube)

HOW DOES INSULIN WORK?

Insulin helps glucose enter the cells, like a key that opens the lock on the cell doors so that glucose, which is blood sugar, enters and is used as energy (Figure 1). If glucose cannot enter because there is no key to open the door, as with people with diabetes, blood glucose builds up. An accumulation of sugar in the blood can cause long-term complications. That’s why it’s important for diabetics to inject insulin.

Figure 1. Picture of the funcioning of insulin in cells (Source: Encuentra tu balance)

WHY DO WE USE TRANSGENIC INSULIN?

First, the insulin obtained from animals such as dogs, pigs or cows was used. But although, above all, pork insulin was very similar to human insulin, it was not identical and contained some impurities. This fact caused rejection and, in some cases, allergies. In addition, to be obtained from the pancreas of pigs, for each pancreas only insulin was obtained for the treatment of 3 days (at more than the cost of care of the animal). The result was low performance and high costs.

But with recombinant DNA insulins, more is obtained at a lower cost. For this reason, currently, the original insulin is obtained from a human of genetic engineering, despite the fact that animal insulins are still a perfectly acceptable alternative.

Through genetic engineering, insulin has been produced from the E. coli bacterium. It was in 1978 when the sequence of the insulin was obtained and introduced inside the bacteria so that it produced insulin. This is how E. coli has gone from being a common bacterium to a factory producing insulin. Insulin is extracted from the bacteria, purified and marketed as a medicine.

The advantages of “human” insulin, obtained by genetic engineering, are the easy maintenance of bacteria, a greater quantity of production and with lower costs. More or more, the compatibility of this insulin is 100%, however there may be reactions due to other components.

On an industrial scale, the production of recombinant proteins encompasses different stages. These stages are fermentation, in which the bacteria are cultivated in nutritious culture media; the extraction to recover all the proteins inside, the purification, which separates the recombinant protein from the other bacterial proteins; and finally the formulation, where the recombinant protein is modified to achieve a stable and sterile form that can be administered therapeutically.

Each of the previous phases implies a very careful handling of the materials and a strict quality control to optimize the extraction, purity, activity and stability of the drug. This process can be simple or more complex depending on the product and the type of cell used. Although the complexity of the process would increase the final cost of the product, the value will not exceed the expense of isolating the compound from its original source to reach medicinal quantities, which is what we have shown with insulin. That is, producing human insulin has a lower cost than obtaining insulin from pigs.

Genetic engineering allows numerous potentially therapeutic proteins to be made in large quantities. Currently, there are more than 30 proteins approved for clinical use, in addition to hundreds of therapeutic protein genes that have been expressed at the laboratory level and that studies continue to demonstrate their clinical adequacy.

REFERENCES

• Ramos, M. et al. El código genético, el secreto de la vida (2017) RBA Libros
• Alberts, B. et al. Biología molecular de la célula (2010). Editorial Omega, 5a edición
• Cooper, G.M., Hausman R.E. La Célula (2009). Editorial Marbán, 5a edición
• Naukas
• Vix
• Main picture: UniversList

Corn as example of genetic improvement in plants

Nowadays, genetic improvement is into the spotlight. However, it is not a new topic because we have done genetic improvement during years, in plants or in animals. In this article, I will discuss genetic improvement in plants, giving the corn as example, which is a plant domesticated by humans for 10,000 years.

WHAT IS GENETIC IMPROVEMENT?

Genetic improvement in plants is the process based on theoretical principles and methods for obtaining varieties of crop plants, which guarantee under high environmental conditions and production, high and stable yields of the products grown with the required quality.

AIMS OF GENETIC IMPROVEMENT

The aims of genetic improvement are:

• Increase performance:
  • Improvement of productivity: increasing the potential productive capacity of individuals.
  • Resistance improvement: obtaining genotypes resistant to pests, diseases and adverse environmental conditions.
  • Improvement of agronomic characteristics: obtaining new genotypes that are better adapted to the demands and application of the mechanization of agriculture.
• Increase the quality: improvement of the nutritive value of the vegetal products obtained.
• Extend the area of exploitation: adapting the varieties of the species already cultivated to new geographical areas with climatic characteristics or extreme soil types.
• Taming new species: transforming wild species into crops with utility and profitability for man.

STEPS OF THE GENETIC IMPROVEMENT PROCESS

Before starting the process, you have to define the objectives you want to achieve and, therefore, define those characters that you want to improve in order to obtain a specific phenotype.

The steps that follow in the process of genetic improvement are:

1. First step: to find within the genetic variability of the collected species, or of the species that can hybridize, individuals that have these characters.
2. Second step: these individuals hybridize with each other and with plants with good general agronomic characteristics. The result will be a base population that will segregate for a large character name, from which individuals will be selected that are closest to the desired variety.
3. Third step: to verify that these individuals are better in one or more aspects than the varieties that are in the market, a fact that normally forces to carry out comparative tests.

THE CORN

The maize plant (Zea Mays) has been domesticated by man for 10,000 years. At this time it has become one of the three most grained cereals in the world and this increase of the crop is linked to the development of varieties that are better adapted to the needs of each place.

Maize is one of the most important staple foods since it makes many derived products (flours, oils). As it has a great value in the industry, it is a much studied plant and its genome has been sequenced.

EUROPEAN CORN BORER

Maize is affected by the European borer (Figure 1), Ostrinia nubilalis. It is a plague of cereals, particularly of corn. It is a native lepidopteran of Europe that infested the millet, before the arrival of the corn.

Its butterfly is about 2.5cm long. The female is yellowish brown with irregular bands on the wings, and the male is smaller and darker. The female lays eggs under the leaves.

Figure 1. Picture of European corn borer female (Source: Discover Life)

The borer makes tunnels inside the corn (Figure 2) that cause the plant to break and fall to the ground. It has to be taken into account that when the maize is still immature it is not affected by the borer, thanks to the natural defenses of these plants in the growing stage.

Figure 2. European corn borer larva in maize (Source: Iowa State University)

GENETIC MODIFICATION OF CORN

We can find two types of modified corn:

• Crops producing their own herbicide (Bt)
• Herbicide-tolerant crops (Monsanto)

Bt maize is a plant genetically modified by modern biotechnology to defend itself against the attack of lepidopteran insects. Using recombinant DNA technology, maize was modified by inserting a bacterium gene of Bacillus thuringensis (Bt), such that its leaves, stem and pollen express the Bt protein of bacteria. Bt maize is the importation and the new tool for the control of damages and losses caused by insect pests.

Herbicide-tolerant maize is maize that has been improved by the use of recombinant DNA technology to tolerate the use of certain types of herbicides. With the use of these technologies for the possible state deactivate or replace the sequence of susceptibility by another that confer resistance and that allow a crop plant to tolerate the use of the herbicide.

OBTAINING THE MAIZE BT

To transform a normal plant to a transgenic plant, the gene that produces a characteristic of interest is identified and separated from the rest of the gene material of a donor organism.

A donor organism can be a bacterium, fungus or any other plant. In the case of Bt maize, the donor organism is a naturally occurring soil bacterium, Bacillus thuringiensis, and the gene of interest produces a protein that kills lepidopteran larvae. This protein is called Bt delta endotoxin.

The Bt delta endotoxin was selected for the fact that it is highly effective for controlling larvae of caterpillars. It is during the larval stage when most of the damage occurs from the European corn borer. The protein is very selective, in general, it does not harm the insects in other orders (like beetles, flies, bees and wasps). Therefore, transgenics that have the Bt gene are compatible with biological control programs, since they harm predators and parasitoids less than insecticides with a broad spectrum of insects. Bt endotoxin is considered safe for humans, other mammals, fish, birds and the environment due to its selectivity.

REFERENCES

• Juan-Ramón Lacadena. Agricultura Transgénica
• Iowa State University – Department of Entomology
• Entomology at the University of Kentucky
• Maíz transgénico: riesgos y beneficios
• Foro en Defensa del Maíz
• Main picture: Informador.mx

How is genetic engineering done in plants?

For years, by crossing, scientists have achieved plants with a desired characteristic after many generations. Biotechnology accelerates this process and allows to catch only the desired genes from a plant, achieving the expected results in only one generation. Genetic engineering allows us to do all this. In this article I will explain what it is and how does it work.

WHAT IS GENETIC ENGINEERING?

Genetic engineering is a branch of biotechnology that consists in modifying hereditary characteristics of an organism by altering its genetic material. Usually it is used to get that certain microorganisms, such as bacteria or viruses, increase the synthesis of compounds, form new compounds or adapt to different environment.

It is a safer and more efficient tool for improving species than traditional methods (crossing) as it eliminates much of the randomness. On the other hand, modern biotechnology also becomes a new technology that has the power to modify the attributes of living organisms by introducing genetic material prepared in vitro.

It could be defined as the set of methodologies to transfer genes from one organism to another and express them (to produce proteins for which these genes encode) in different organisms of the original organism. DNA which combines fragments of different organisms is called recombinant DNA. Consequently, genetic engineering’s techniques are called recombinant DNA techniques.

Currently there are more plant organisms genetically modified than animal organisms. For this reason I will explain genetic engineering based on plants.

GENETIC ENGINEERING vs. TRADITIONAL METHODS

This methodology has 3 key advantages compared with traditional methods of genetic improvement based on hybridization:

• The genes could come from any specie (for example a bacteria’s gene can be incorporated in soy‘s genome).
• At genetically improved plant you may introduce a single new gene preserving the remaining genes from the original plant to their offspring.
• This modification process delays less the deadlines than improvement by crossbreeding.

With this way you can modify properties of plants more broadly, more accurate and faster.

In traditional crossing it generates a hybrid which combines randomly genes of both parental organisms, including the gene of interest encoding the desired trait. In contrast, biotechnology techniques only pass one or few genes which encode a specific trait known. The new plant has all the original genes of the plant and an introduced gene accurately and directed (Figure 1).

Figure 1. (A) Traditional method where, by crossing, a new variety is obtained. This carries the gene of interest (red), but also another genes randomly. (B) With genetic engineering we obtain a new variety of commercial plant with the gene of interest (red) of any other species (Source: Mireia Ramos, All You Need is Biology)

METHODOLOGY OF GENETIC ENGINEERING

Obtaining a transgenic organism through genetic engineering techniques involves the participation of an organism who gives the gene of interest and a receptor organism who will express the desired quality. The steps and the process techniques are:

0/ DECIDE THE AIM: MAKE KNOCK IN OR KNOCK OUT

KNOCK OUT:

This technique is to remove the expression of a gene, replacing it with a mutated version of itself, this being a non-functional copy. It allows the gene is not expressed.

KNOCK IN:

It is the opposite of the knock out process. A gene is replaced by a modified version of itself, which produces a variation in the resulting function of it.

In medicine, the knock in technique has been used as a strategy to replace or mutate genes that cause diseases such as Huntington’s chorea, in order to create a successful therapy.

1/ DOUBLE CHECK THAT THERE IS A GENE CODING FOR THE CHARACTERISTIC OF INTEREST

Firstly, you have to check the characteristic of interest comes from a gene, as this will be easier to transfer to a living organism that does not.

2/ CLONING THE GENE OF INTEREST

It is a complex process, but outline the steps are the following:

• Extract DNA
• Find a gene among the genes of this DNA
• Sequence it
• Build the recombinant vector

The DNA of interest is inserted into a plasmid, a circular DNA molecule with autonomous replication. The plasmids of bacterial origin are the most used (Video 1).

Video 1. “Clonación de un gen en un plásmido vector”. Explaining the use of plasmids as a vector in the process of cloning (Source: YouTube)

The development of these techniques was possible by the discovery of restriction enzymes. These enzymes recognize specific sequences and cut the DNA by these points. The generated ends can be sealed with ligase enzyme and to obtain a new DNA molecule, it called recombinant DNA (Figure 2).

Figure 2. (1) Plasmid’s DNA (2) DNA from another living organism (3a, 3b) The restriction enzyme cuts DNA (4) The restriction enzyme recognizes AATT sequence and cuts between A and T nucleotides (5) The two DNAs are contacted with the purpose of forming recombinant molecules (6) A ligase enzyme joins the DNA ends (Source: GeoPaloma)

3/ CHARACTERISE GENE OF INTEREST

If we know the gene sequence we can compare this sequence with known gene sequence through bioinformatics, provided to determine which gene looks and assign a possible function. So when we have predicted the function of cloned gene we confirm it in vivo, usually transferring it to a model organism.

4/ MODIFY GENE OF INTEREST

We can add (promoter, introns…) or mutate sequences inside the encoding region.

5/ TRANSFORMATION OF A LIVING ORGANISM WITH GENE OF INTEREST

When we have finished the gene building with the desired gene and the promoter, the recombinant DNA is inserted into the cells of the living organism that we want to modify.

6/ CHARACTERIZATION GMO

When we already have the GMO (Genetically Modified Organism) it is analysed from the molecular and biological point. In the molecular analysis it must demonstrate if you have one or more copies of the transgene or how and what tissues the gene is expressed. In the biological analysis it looks if it achieves the objective for which it was designed.

REFERENCES

• Por qué biotecnología
• Cultivos Transgénicos: Introducción y Guía a Recursos
• Agricultura Transgénica
• Main picture: FarmacoSalud