Understanding Molecular and Cellular Cloning Techniques
Molecular Cloning
Molecular cloning is used in a wide variety of biological experiments and practical applications, ranging from fingerprinting to large-scale protein production. In practice, to amplify any sequence in a living organism, the sequence to be cloned must be linked to an origin of replication, which is a DNA sequence.
Key Steps in Molecular Cloning:
- Transfection: Introduces the formed sequence within cells.
- Selection: Selects the cells that have been successfully transfected with the new DNA.
Initially, the DNA of interest needs to be isolated from a DNA segment of suitable size. Subsequently, the ligation process occurs where the amplified fragment is inserted into a cloning vector. The vector is linearized (as it is circular) using restriction enzymes and then incubated under appropriate conditions with the DNA fragment of interest and the enzyme DNA ligase.
Following ligation, the vector with the insert of interest undergoes transfection within cells. For this purpose, transfected cells are cultured. This is a crucial step, as it determines whether the cells have been successfully transfected or not.
We must, therefore, identify the transfected and non-transfected cells. Modern cloning vectors include markers of resistance to antibiotics, which only cells that have been transfected can grow. Other cloning vectors provide blue/white screening. Thus, the investigation of the colonies is needed to confirm that cloning was successful.
Cell Cloning
Cloning a cell involves forming a group of cells from a single one. For unicellular organisms like bacteria and yeast, this process is very simple and only requires the inoculation of the right products.
However, in the case of cultured cells in multicellular organisms, cell cloning is a difficult task because these cells need very specific environmental conditions.
A useful tissue culture technique used to clone distinct lineages of cells is the use of cloning rings (cylinders).
According to this technique, a group of unicellular cells that have been exposed to a mutagenic agent or drug used to promote selection is placed in a high dilution to create isolated colonies, each coming from a single and potentially clonally distinct cell.
In the first stage of growth, when the colonies have only a few cells, sterile polystyrene rings are dipped in fat and placed over a single colony with a small amount of trypsin.
The cloned cells are collected in the ring and transferred to a new container to continue their growth.
Therapeutic Cloning
Therapeutic cloning has therapeutic purposes and involves obtaining stem cells from the patient to be treated. Consider the following experiment: A somatic cell is taken from the patient to be treated. The nucleus with the chromosomes is isolated, and everything else is discarded.
On the other hand, an unfertilized egg is obtained, and its nucleus with its chromosomes is extracted. The previously isolated nucleus of the somatic cell is then introduced into this egg. The egg with the nucleus is stimulated, and cell division starts from the cloned embryo.
This embryo is a clone of the patient to be treated. The embryo is allowed to develop until it reaches a key stage: the blastocyst.
At this stage, stem cells are extracted from the cell mass, which has the same DNA as the patient and, therefore, will not cause rejection when injected.
An example of this type of cloning is the cloning of Dolly (July 5, 1996 – February 14, 2003).
Cloning for Stem Cell Research
Somatic cell nuclear transfer can also be used to create a cloned embryo. The goal is not to clone humans but (as mentioned previously) to harvest stem cells that can be used to study human development and undertake research on diseases of interest.
Cloning of Naturally Occurring Organisms
The cloning of an organism is to create a new body with the same genetic information as an existing cell. It is a method of asexual reproduction, where fertilization does not occur. Overall, only one parent is involved. This form of reproduction is common in organisms such as amoeba and other unicellular organisms, although most plants and fungi also reproduce asexually.
Also included is obtaining identical twins naturally or artificially. The natural form is considered a spontaneous alteration during embryonic development, ignoring their cause, although a statistically significant correlation exists within families. The artificial method is performed by manipulating the blastomeres, weakening the cell junctions with a poor medium with trypsin in Ca2+, or manually starting the blastocyst in the middle (very common in cows).
Human Cloning
Human cloning is the creation of a genetically identical copy of a current or former human being. There are three types of human cloning:
- Andropatrica Cloning
- Reproductive Cloning
- Hydroplasmotica Cloning
Andropatrica cloning involves cloning cells from an adult individual for subsequent use in medicine (as seen in the section on therapeutic cloning).
Reproductive cloning would involve the complete cloning of a human being. This type of cloning has not yet been done in humans.
Hydroplasmotica cloning involves the configuration of cloning humans using a mechanism that is hydropower.
A fourth type of cloning would be replacement cloning, which would be a combination of reproductive cloning and therapeutic cloning. In this type of cloning, the partial cloning of a tissue or part of a human needed for a transplant would be produced.
In January 2008, it was announced that five human embryos were created using the DNA of skin cells from adults to provide a viable source of stem cells. Using the same technique that led to Dolly the sheep, scientists at the Californian company Stemagen Corporation (based in La Jolla, CA), led by Andrew French, used skin cells from two adult males and the eggs of three young women (between 20 and 24 years old) who were undergoing fertility treatment. One skin donor was Samuel Wood, executive director of the company and co-author. However, ethical and legal concerns were raised, so they were destroyed.[2]
The purpose of human cloning research has never been to clone people or create reserve babies.[3] The research aims to obtain stem cells to cure diseases.[4]
Of course, the results of research on animal and human cloning to obtain stem cells have been published, and, like other scientific discoveries, these publications are available worldwide.
These individuals are not employed by any university, hospital, or government institution. In general, the worldwide scientific community strongly opposes any suggestion to clone a baby.
According to John Kilner, president of the Center for Bioethics and Human Dignity in the United States, “The majority of published research shows that the death or maiming of the clone are very likely results in the cloning of mammals.”
Nobody knows how much has actually gone into human cloning babies. In April 2002, Italian scientist Dr. Severino Antinori made an impromptu comment to a reporter, saying that three women were pregnant with a cloned embryo. Since then, he has departed from under the stage lights and never had the opportunity to confirm or deny this comment. Although not true, or the attempt had failed, it gave the impression that Antinori intended to try to clone a human baby in the near future.
Doctors rate the risk of human cloning as very high.
“Yielding to the cloning of humans does not mean assuming an unknown risk, but consciously hurting people,” says Kilner.
Most scientists are of the same opinion. The vast majority of attempts to clone animal embryos resulted in distorted embryos or abortions after implantation. They argue that the few cloned animals born have malformations not detectable by analysis or tests in the uterus, for example, strains in the lining of the lungs.
In 1996, Dolly the sheep was cloned. It was the first mammal cloned from DNA derived from an adult instead of using DNA from an embryo. But while Dolly has a healthy appearance, there are questions about the possibility that she aged faster than a normal sheep. In addition, it took 277 embryos to produce this birth.