Copy-paste Life: The Science of Animal Cloning

Cloning, as described by Linn et al. in 2013, is the creation of genetically similar organisms. This can occur through the splitting of an embryo, either by duplicating nuclear genes or some mitochondrial genes, giving rise to what we refer to as "identical" organisms. The word "clone" was originally coined by Herbert in 1903 from the ancient Greek word "klon," meaning twig, similar to how a new plant can grow from a twig. Cloning is a natural reproductive process that has allowed different life forms to exist for hundreds of millions of years and is frequently employed by plants, fungi, and bacteria. Cloning has been applied for a variety of purposes, ranging from the rapid reproduction of valuable animal breeds, the generation of transgenic animals, purposeful genetic alterations in domestic animals, and even the conservation of endangered animals. Although cloning is generally used for the replication of DNA fragments carrying whole genes, it can be employed to amplify any DNA sequence, including promoters, non-coding regions, and randomly sheared DNA. Its flexibility renders it a precious asset in many fields of biotechnology.

There are examples of cloning all around us, the case of identical twins being the most obvious, even without counting the forms of cloning found in plants and invertebrates, Scientists started studying artificial cloning at the turn of the last century when a German scientist, Hans Adolf Eduard Driesch, began researching reproduction in salamanders. In 1902, he split an embryo into two separate viable embryos.  Further research on frogs in the 1950s and 1960s showed that differentiated somatic cell nuclei could be dedifferentiated into embryos. In the 1970s, animal scientists adapted techniques like embryo splitting and blastomere cloning to improve production efficiency and genetic gain. Some of the early results were controversial, partly due to experimental reproducibility, but it continued to grow the field of animal research. The biggest breakthrough came in 1996 when researchers at the Roslin Research Institute in Scotland cloned the first live lamb known as Dolly. Dolly was the first mammal to be cloned from the somatic cells of an adult animal. Studies have revealed that it took 277 attempts until researchers finally cloned Dolly, which survived longer than most other cloned lambs. Her cloning opened up a new agricultural era, with possibilities of protecting and duplicating individual genotypes, and the success of Dolly made researchers think of new ways of modifying cells. Since Dolly, more animal clones have been produced, and even human cells have been experimented on. Stem cell research has even reached a point where it’s being used for therapeutics like hair growth and burn patients.

Types of Cloning

Reproductive Cloning

• This procedure entails the insertion of a cloned embryo into either a natural or artificial uterus. The embryo grows to become a fetus, which is then carried to term.

• The methods for reproductive cloning experienced a significant transformation during the 1990s, particularly following the birth of Dolly, the world's first cloned sheep, born by somatic cell nuclear transfer (Colman, 1999).

• Reproductive cloning entails removing the nucleus from a somatic (body) cell and placing it into an egg cell that has had its nucleus removed (a procedure referred to as enucleation). After the somatic nucleus is within the egg, a mild electrical current is used to induce division, creating a cloned embryo that is a genetic duplicate of the original organism.

Therapeutic cloning

• Therapeutic cloning involves the use of cloned embryos for the extraction of stem cells without implanting the embryos into a womb. It enables us to culture stem cells that are genetically matched with the patient. These stem cells can be stimulated to differentiate into more than 200 various cell types present in the human body. After differentiation, these cells are then able to be transplanted back into the patient to replace ill or injured cells without evoking an immune response.

• The method holds great potential to cure several diseases and disorders, including Alzheimer's disease, Parkinson's disease, diabetes, strokes, and spinal cord injury.

  
  

Cloning Techniques

Male Pronuclear Microinjection

• The microinjection of male pronucleus with naked DNA is the first technique successfully utilized to obtain transgenic animals.

• In this method, eggs (oocytes) are harvested from super-ovulated (matured) animals and fertilized in-vitro. A microtube is used to hold the fertilized egg in place, and an extremely fine needle is used to inject a tiny amount of solution, which contains many copies of the foreign DNA (transgene), into the male pronucleus. These eggs are then inserted into the surrogate mother’s oviducts.

• It is currently the main method for creating genetically altered animals, involving the actual injection of 200-300 copies of the foreign gene into the surrogate mothers.

Sperm-Mediated Gene Transfer (SMGT)

• This technique relies on the intrinsic ability of sperm cells to bind and internalize exogenous DNA and to transfer it into the oocyte at fertilization.

• This technique is the first optimizer for application in small laboratory animals, showing high efficiency for mice.

• SMGT has been successfully adapted and optimized for use in large animals.

• The technique has found its use in producing Human Decay Accelerating Factor (HDAF) transgenic pig lines with high-efficiency transgenic pigs showing good protection against hyperacute rejection in ex-vivo experiments.

Applications of Cloning

Rapid reproduction of desired livestock

• Cloning enables the rapid dissemination of superior genotypes from nucleus-breeding flocks and herds directly to commercial farmers.

• Genotypes can be supplied which are best suited to specific product characteristics, disease resistance, or for environmental conditions.

• Cloning could be extremely useful in multiplying outstanding F1 crossbred animals, or composite breeds, to maximize the benefit of both heterosis and potential uniformity within the cloned family. 

Conservation of animals

• Cloning can be used along with other forms of assisted reproduction to help preserve indigenous breeds of livestock, which have production traits and adaptability to local environments that should not be lost from the global gene pool.

• Cloning can be used to recover extinct species, as long as some of their cells are saved through freezing. This choice is a strong incentive to maintain tissue banks of threatened species of animals.

• Cloning is a potential way to rescue threatened species. In January 2009, researchers at the Centre of Food Technology and Research of Aragon, in Zaragoza, reported the cloning of the Pyrenean ibex, a species of wild mountain goat, which had been announced as extinct in 2000. Using the DNA from skin samples preserved in liquid nitrogen, the researchers cloned the ibex from domestic goat egg cells. The newborn ibex died shortly after its birth due to physical deformities in its lungs. However, it is the first time an extinct species has been cloned, and it may open up ways to save endangered and recently extinct species by reviving them from frozen tissues.

Research models

• A set of cloned animals could be effectively used to reduce genetic variability and reduce the number of animals needed for experimental studies. This could be conducted on a larger scale than is currently possible with naturally occurring genetically identical twins, e.g., a lamb cloned from sheep selected for resistance or susceptibility to nematode worms will be useful in studies aimed at discovering novel genes and regulatory pathways in immunological studies.

Human cell-based therapies

• This is the direct use of nuclear transfer technology in human medicine, mainly in the field of therapeutic cloning.

• Patients with specific diseases or disorders of tissues that do not repair or replace themselves well (e.g. insulin-dependent diabetes, muscularly dystrophy, spinal cord injury, certain cancers, and other neurological diseases, such as Parkinson's disease) might produce their own immunologically compatible cells for transplantation, which would provide lifelong therapy without tissue rejection.

Xeno-transplantation

• Xeno-transplantation refers to the repositioning of cells, tissues, or organs from one species to another, such as from pigs to humans.

• The fact that pig organs are more analogous to those in humans in terms of anatomical structure and physiological and biochemical characteristics makes them suitable for xeno-transplantation than most primates. In addition to the selection of pigs as the preferred donors, an additional significant and most important advantage is the ability of pigs to get genetically modified.

  
  

Ethical Issues in Cloning

Animal Welfare Concerns

• Low success rate

  Cloning attempts often fail, with low success rates, especially during development. 

• Expensive and time-consuming

  Cloning is costly, complex, and laborious, not practical for wide use.

• Suffering during the process

  Due to the invasive nature, animals experience high stress levels during cloning procedures, which result in numerous unsuccessful outcomes and health complications affecting both the mother and the cloned offspring.

• Reduced genetic diversity

  A diminished genetic diversity following animal cloning procedures would make existing populations of animals more susceptible to health risks and environmental changes.

• Health problems in cloned animals

  Cloning as a method leads to health complications, which include growth problems referred to as "large offspring syndrome" along with developmental problems and heightened disease vulnerability.

• Unanticipated consequences

  Future health problems remain unknown for cloned animals, so additional detrimental outcomes may materialize.

Moral and Ethical Concerns

• Interference with nature

  Through the scientific procedure of cloning, scientists are attempting to control natural evolutionary methods.

• Intrinsic value of animals

  Animals are devalued when treated as objects instead of valued beings through the cloning process.

• Co-modification of animals

  When animals are cloned for particular purposes, there is a risk that this will generate greater animal neglect and exploitation.

• Slippery slope to human cloning

  The ethical debate rises about animal cloning because some speculate it will lead to human reproductive cloning, thus creating additional moral dilemmas.

• Unintended consequences for the environment

  The dominance of clones from a single breed could cause harm to ecosystems while simultaneously decreasing animal species diversity in populations.

• Deception and exploitation

  The business of pet cloning faces criticism due to companies who might take advantage of pet owners’ grief through false promises to return deceased pets.

  
  

Way Forward

Enhancing cloning techniques like Somatic Cell Nuclear Transfer (SCNT) and gene editing can improve success rates and safety. Therapeutic cloning offers potential treatments for diseases such as Parkinson’s and Alzheimer’s. Ethical frameworks are essential to regulate cloning responsibly. Cloning can aid in species conservation, while public engagement and policy frameworks are key for addressing concerns and informed governance.

Conclusion

Cloning holds great potential for medicine, conservation, and research but faces technical, ethical, and regulatory challenges. Advances in therapeutic cloning may lead to breakthroughs in disease treatment, while reproductive cloning could aid biodiversity efforts. However, responsible governance, public engagement, and ethical frameworks are essential to balance innovation with societal concerns, ensuring safe and beneficial outcomes.

References

1. Bordignon, V. (2017). Animal Cloning – State of the Art and Applications. Reference Module in    Life Sciences. doi.org/10.1016/b978-0-12-809633-8.09221-9. Accessed on 21/9/2021.

2. Colman, A. (1999). Somatic cell nuclear transfer in mammals: progress and application.     Cloning, 1(4): 185-200.

3. Garry, F.B., Adams, R., McCann, J.P. and Odde, K.G. (1996) Pre and postnatal characteristics of calves produced by nuclear transfer cloning. Theriogenology, 45(1):141-152.

4. Hall, J.G. (1996). Twinning: mechanisms and genetic implication. Current Opinion in Genetic   Development, 6(3): 343-347.

5. King, W., Yada, R. and Grodzinski, B. (2011). Principles of cloning. Comprehensive  Biotechnology, 12(354): 781-785. 

6. Linnaeus, C.Y. and Solter, D. (2013). Cloned organisms: Eukaryotic. Brenner’s Encyclopedia of  Genetics, 6(12):46-48.

7. Wells, D.N. (2003) Immune status: normal expression of MHC in placenta and what is expected in clones. Cloning and stem cells, 6(2): 121-125.

8. Wolf, E., Zakhartchenko, V. and Brem, G. (1998). Nuclear transfer in mammals: recent developments and future perspectives. Journal of Biotechnology, 27(65): 2-3: 99-110.