Arxiu d'etiquetes: clone

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).

fig1ENG
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).

adnrecombi
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

MireiaRamos-angles

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Immaculate Conception… in reptiles and insects

December’s bank holidays and Christmas’s holidays have in common in that the Immaculate Conception is celebrated in both. The biological phenomenon in which a female animal reproduces without mating with a male is called parthenogenesis and, even if there isn’t any proof that this could happen to human beings, virginal birth is a widely distributed thing throughout the animal kingdom. In this entry we’ll see how this incredible phenomenon happens and some species in which it appears.

WHAT IS PARTHENOGENESIS?

Parthenogenesis is a type of asexual reproduction in which the offspring comes from a non-fertilized ovum. Without fertilization (union of the oocyte’s and the sperm’s genetic material) the offspring won’t have any part of the father’s DNA (if there is a father). The resulting babies will be genetic copies (clones) of their mother.

532px-Haploid,_diploid_,triploid_and_tetraploidDuring fertilization, when the ovum and the sperm fuse together (both haploid cells, with just one copy of chromosomes, n chromosomes) a new individual is formed with a unique genetic combination, with DNA from its father and its mother (diploid, with two copies of each chromosome, 2n chromosomes in each cell). Triploid (3n) or tetraploid (4n) individuals only appear in asexual hybrid species, and most cases are non-viable. Images by Ehamberg.

In parthenogenetic animals, the lack of paternal genetic material must be compensated because in many species haploid foetuses are non-viable. In these species diploidy (2n chromosomes) is usually re-established through a process called automixis. Yet in some species, haploid individuals with parthenogenetic origins are viable and have no problems in surviving.

It is impossible to pose a general example for asexual reproduction, as it is widely distributed through very different animal groups and there are many cases with many differences among them. Bellow, we’ll present you some examples of different strategies used by animals to reproduce asexually.

HAPLODIPLOIDY IN BEES AND WASPS

Haplodiploidy is a phenomenon that appears in two insect orders, hymenopterans (bees, ants and wasps) and thysanopterans (thrips or stormbugs). In this sexual determination system, if the ovum is fertilized it will develop into a female while, if it isn’t fertilized a haploid male will be born.

Apis_Mellifera_Carnica_Queen_Bee_in_the_hiveColony of Carniolan honey bees (Apis mellifera carnica), a subspecies of hony bee from Eastern Europe. Photo by Levi Asay.

In the honey bee, when the queen bee mates with a drone (male bee), all the diploid individuals (2n) will became females, with DNA combined from the queen and the drone. By contrast, drones are born by parthenogenesis, in which an egg from the queen will develop into a haploid drone (n). This means that the individuals in a bee colony, descendants from the same queen, are much more closely related to each other than regular siblings (drones have 100% of their mother’s DNA). It is believed that this helped to the development of eusocial behaviours in different hymenopteran groups.

CYCLIC PARTHENOGENESIS

This kind of parthenogenesis is found in different invertebrate groups that can alternate between asexual and sexual reproduction during its life cycle depending on the environmental conditions.

1471-2164-14-412-1-lDiagram about the life cycle of a rotifer, in which parthenogenetic asexual reproduction during good environmental conditions is alternated with sexual reproductions with a haploid male during adverse conditions. Image extracted from Hanson et al. 2013.

Some invertebrate groups like aphids, present asexual parthenogenic reproduction from spring until early autumn, when conditions are favourable. During this stage in many populations we find only females that give birth to more females.

Fast motion video in which we can see how the aphids take advantage during good weather conditions to increase fast and efficiently the number of individuals asexually. Video by Neil Bromhall.

When autumn approaches, parthenogenetic females start giving birth to sexual males and females. Both sexes are born by parthenogenesis and have 100% of their mother’s DNA. Sexual winged individuals then disperse to avoid mating with their own siblings. These will mate and females will lay resistant eggs that will survive winter. In spring these eggs will hatch and give rise to a new generation of parthenogenetic females that will start the cycle again.

TRUE PARTHENOGENESIS IN SQUAMATES

The only vertebrates that show true parthenogenesis are the squamates, with about 50 lizard species and one snake being obligate parthenotes. These are unisexual species, all individuals being females that reproduce asexually without the intervention of any male. Also, there are many other species that, even if they usually reproduce sexually, are also able to reproduce asexually when there are no males available (facultative parthenogenesis).

DesertGrasslandWhiptailLizard_AspidoscelisUniparensDesert grassland whiptail lizard (Cnemidophorus uniparens) which, as its scientific name implies, is a parthenogenic species in which all specimens are female. Photo by Ltshears.

There are isolated cases of captive female sharks, snakes and Komodo dragons that have reproduced without fertilization or mating with a male. Yet, this is known as accidental parthenogenesis, because the high mortality of the offspring (surviving between 1/100.000 and 1/million) shows that it is probably due to a failure of the organism, more than an adaptive phenomenon.

ParthkomodoBaby Komodo dragon (Varanus komodoensis) born by accidental parthenogenesis at Chester Zoo. Photo by Neil.

Females from the true parthenogenetic species produce haploid eggs (with n chromosomes) which eventually become diploid (2n chromosomes) by two consecutive division cycles during meiosis (automixis). In species with facultative parthenogenesis, diploidy is achieved by the fusion of the ovum with a haploid polar body that forms during meiosis.

Oogenesis-polar-body-diagramScheme of the formation of polar bodies during oogenesis, which may help parthenogenetic reptiles to regain their diploidy. Scheme by Studentreader.

True parthenogenesis is especially well-known in the Brahminy blind snake (Ramphotyphlops brahminus) and many species of lizards. In these species females generate clones of themselves. Parthenogenetic lizard species (like in amphibians) probably originated from a hybridization event between two sexual species. Many whiptail lizards (genera Cnemidophorus/Aspidoscelis) present unisexual species in which no males exist, from a hybridation process.

Ramphotyphlops_braminus_in_Timor-LesteBrahminy blind snake (Ramphotyphlops braminus), the only known unisexual ophidian, in which all specimens found to date are females. Photo taken from Kaiser et al. 2011.

The species Cnemidophorus uniparens is a parthenogenic unisexual species, which appeared asa result of the hybridization between C. inornatus and C. burti. The resulting hybrid reproduced again with C. inornatus, forming the triploid (3n) parthenote C. uniparens. The presence of triploid, tetraploid, etc. genomes is a common phenomenon between unisexual reptiles, as its hybrid origins sometimes prevents the mixing of genomes. Also, a greater chromosomal variability compensates the lack of genetic recombination.

Despite being unisexual, sexual behaviours have been observed in this species similar to bisexual species. In C. uniparens there are documented sexual behaviours in which one female takes the role of a male and “mounts” another female contacting their cloacae. It is known that mounted females increase their egg production after this fake copula. It is believed that from one year to the other females shift their roles of mounting or being mounted, varying from year to year the number of eggs laid.

Cnemidophorus-ThreeSpeciesThree species of whiptail lizards. The middle one, Cnemidophorus neomexicanus is an unisexual parthenogenic species, originated from the hybridization of two bisexual species, C. inornatus (left) and C. tigris (right). Photo by Alistair J. Cullum.

Even if they are true parthenogenetic species, many of these squamates keep their ability to add new DNA to their offspring. This is due to the fact that if there’s no genetic recombination by the fusion of the ovum and the spermatozoon, there’s a high risk of accumulating genetic mutations detrimental for the species. Yet parthenogenesis allows these species to quickly colonize new habitats, because it is not necessary for two individuals to find each other to procreate, and 100% of the population is able to reproduce.

As you can see, there is a great number of animals that don’t need males nor sex to reproduce. The existence of a similar process in human beings is pretty much improbable (no to say impossible). Besides, if 2000 years ago a woman would have given birth to a baby without fertilization, probably this would have been a girl, because it wouldn’t have been able to acquire the Y chromosome from anywhere. Yet, this doesn’t mean we cannot enjoy the upcoming holidays. Merry Christmas and Happy New Year to everyone!

REFERENCES

The following sources have been used during the elaboration of this entry:

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