Cloning to Save a Species

How could cloning an individual organism save the species?

When we typically think about a clone, we imagine identical organisms that are exactly the same as the DNA donor such as examples from popular fiction movies: Gemini Man, Replicas, and The 6th Day. To an extent, a cloned organism can be said to be genetically the same but with new methodologies used in recent studies even that is debatable. However, even if we agree a clone is a genetically identical, one could wonder how creating more of the same organism could save a species.


Imagine a critically endangered population coming down to the last dozen of their species, 6 males (M) and 6 females (F) in a perfect situation. How much longer can the population last breeding between these dozen individuals before inbreeding their traits?


Figure 1 shows the offspring potential if each male mate with each female and produce only 1 offspring.


Figure 1. 6X6 Single Offspring Potential

This initially may look like a lot of breeding possibilities, especially since many species create many offspring within one breeding season or breed many times within one life span. Let us take a closer look at the offspring breeding potential. Since we are trying to prevent inbreeding to promote genetic diversity and prevent inbreeding deformities and complications, offspring within the same rows and columns cannot mate together because they contain one of the same parents.


We have developed the technology to be able to obtain and store cells from individuals from endangered populations preserving their DNA due to the Frozen Ark Project [1]. With this advancement we can preserve the genetic diversity of wild individuals within decimating species before they reach such low numbers and reintroduce clones of the wild individuals that were lost [1]. This method could increase genetic diversity into dwindling populations that are struggling to find viable mates.


For example, suppose we have a small population of endangered rhinos and are working on a breeding program to sustain their population. Male #1 (1M) failed in competition to fight for mating rights to secure a female mate. Due to his insufficient horn size he was severely injured in the fight and died, failing to reproduce in this generation. All his potential progeny has been marked red. Female #1 (1F) was sick with an infection. Poachers hunted and killed her to acquire her horn for ivory sales. She failed to give birth before her death, so all her potential progeny has been marked red. A new female clone has been introduced that was made for the cells acquired from a deceased rhino found after being hunted. This new female adds genetic diversity to the mating pool and recovers mating potential that was lost from unexpected events in the breeding program as shown in figure 2.


Figure 2. Exclusion of Selected Individuals and Clone Recovery

With this model in mind, cloning serves as a prospective preventative measure to maintain genetic diversity in small populations of keystone species in an ecosystem decimated by human impacts that otherwise would have maintained stability [1]. Without introducing cloned variants, distanced inbreeding models for smaller populations leave the species more likely to acquire harmful genetic mutations, diseases, or losses due to weaker trait heredity [1]. If we act early to develop and preserve DNA banks for the endangered populations, cloning may stand a chance to help us build and recover a species in sustainable numbers alongside of conservation practices to mitigate hunting, habitat destruction, and other human impacts.


How does SCNT cloning happen? What does the process look like?


Cloning complex organisms has been accomplished with leopard frogs, the infamous Finn Dorset sheep named Dolly, mouflon sheep, gaurs, buntings, the Pyrenean ibex, and coyotes. The processed used to clone sexually reproducing higher order organisms is called somatic cell nuclear transfer or SCNT [2]. This process requires a viable DNA from a somatic (body) cell of the clone species, an egg to implant the DNA, and a womb or incubating conditions for embryonic development [2]. The step process is simplified and outlined in figure 3.



Figure 3. SCNT Process Outlined


What issues have arisen using SCNT to save endangered species?


When the goal is to conserve a species with long term sustainability, retaining future reproductive success, limiting cellular trauma, and maintain functioning in the wild are important variables to consider. To successfully perform SCNT on a species high numbers of embryos need to be constructed and high numbers of pregnancies must be studied to successfully produces just a few clones [3]. An understanding of the reproductive system is critical which is a limiting factor for scientist in obtaining the number of eggs from a species and limiting the ability for scientist to use the same species to perform the pregnancy [3].


When critically endangered species fall to severely dwindling numbers, legislation protects the species from the egg extractions and tissue collecting procedures. To work around this, scientist have used eggs and wombs for surrogacy of closely related species with limited success and development obstacles [3]. The hybrid embryos with mitochondrial DNA of another species egg has it's DNA instructions reprogrammed with another species instructions [3]. This becomes an issue in the embryotic development of some species however limited success has been achieved in isolated studies [4].


Cloning Species With Limited Eggs


Since the mitochondrial DNA override poses high uncertainty with the egg development to viable embryos, scientists will collect multiple eggs from a different species. The viable embryos from the fused egg and donor somatic cell DNA will be clustered together to improve embryotic development [5]. In prior studies that tested this technique from species categorized in a different genus, no effects were observed [5]. However, a recent study that tested this technique with Yak DNA categorized in the same genus as the egg donor, the development was improved [5].


Frozen vs Living Cells


To successfully clone sexually reproducing complex organisms, the scientist must obtain undamaged nuclei from usable cells [6]. The process works best when the cells are isolated from tissues of a living donor however deceased animals are often frozen which causes damage to cell parts, organelles, from water crystals that form within the cellular structure [6]. A study performing SCNT on five deceased frozen canines without cryoprotectant was performed [6]. The study noted growing cells from frozen tissues from the deceased canines required a longer period than living tissue sources [6]. The study also noted that the longer the tissue is frozen, longer time is required to grow the cells for the process [5]. Death cells not viable for the process are higher in frozen cells than living cells and higher the longer the cells are frozen [6]. Ultimately, the cloning process and pregnancy rates are lower in deceased frozen cells than cell from living donors or recently frozen living cells [6].


Livestock germplasm being frozen in liquid nitrogen (Photo Credit: USDA Gene Bank / CC0)

Cloned Embryos vs Fertilized Embryos


The entire idea of cloning is founded on the understanding that the nucleus of one cell in your body contains the exact same DNA as other cells in your body. The difference is that different cell types express different parts of the DNA sequence. However, scientist wondered if the nucleus of the body cell has a significant difference from the nucleus from the egg that is fused from the fertilization of sperm. What we really want to know is what the process of cloning is missing from lacking the fusion of sperm in sexual reproduction. A study analyzed fertilized embryos and embryos of SCNT cloned embryos [7]. The SCNT cloned embryos were lacking the sperm noncoding RNA and proteins [7]. The study determined that missing these molecular components could be a cause for the development obstacles observed in SCNT embryos [7].

Another study analyzed the specific gene expression differences through in vitro fertilized (IVF) embryos and SCNT cloned embryos [8]. The study detected that 356 genes showed less expression in cloned embryos and 593 genes were less expressed in clones in the early blastocyst stage of development [8]. Researchers noted 14 genes related to the development of the cloned embryos to be inactivated and 68 noncoding RNA sequences were found to be irregularly expressed in cloned embryos [8]. Scientist also reported that the RNA editing that occurred in cloned embryos were incompletely executed which may be the reason for a specific gene to have less gene expression [8].


In Vitro Fertilization (Photo Credit: Dr. Kontogianni)



Potential Use of Cloning for De-Extinction


Researchers have begun attempts on cloning the Woolly Mammoth from recovered tissue. The frozen tissue obtained from the long extinct organism provides many obstacles for the current state of cloning. We now know that the damage to the nucleus from water crystallization will require reconstruction to the genome. Scientist have attempted hybridizing the Woolly Mammoth DNA with an elephant species containing a common ancestor. CRISPR technology proves to be a service in advancing the research to construct a viable complete genome to implant in the eggs of modern species [9]. Then the challenge of advancing viable embryos in surrogacy to term births will prove another challenge. Scientist have already successfully cloned the Spanish goat although the lifespan of the clone did not last beyond a few minutes [9].


There are many variables to consider before this prospect of using SCNT for de-extinction comes to fruition. What complications can we expect in the surrogate carrying the hybrid? Will the Woollyphant or Elemamoth hybrid be able to survive in today’s climate? Will the species be allowed to be introduced to the wild and how could the introduction impact ecosystems? Will the species be able to breed and how could it impact the survival and preservation of modern elephant species? Only further advancing the research will reveal the answers to these questions. We are more likely to see the remnants of the Woolly Mammoth traits genetically engineered into a modern elephant species [10].


References


1. Clarke, A. (2004) Frozen Ark Project. Retrieved from www.frozenark.org

2. Liu L. (2020) Nuclear Transfer and Cloning. In: Larson M. (eds) Transgenic Mouse. Methods in Molecular Biology, vol 2066. Humana, New York, NY

3. Wang, X., Qu, J., Li, J., He, H., Liu, Z., and Huan, Y. (2020). Epigenetic Reprogramming During Somatic Cell Nuclear Transfer: Recent Progress and Future Directions. Retrieved from https://doi.org/10.3389/fgene.2020.00205

4. Hiendleder, S., Zakhartchenko, V., Wolf, E., (2005). Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear–mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination. Reproduction, Fertility and Development 17(2) 69-83. Retrieved From: https://doi.org/10.1071/RD04115

5. Felipe, M. Y., Rodríguez, M.D., De Stéfano, A., & Salamone, D. (2019). 20 Aggregation of yak heterospecific somatic cell nuclear transfer embryos improves cloning efficiency. Reproduction, Fertility and Development 32(2) 135-136. Retrieved From: https://doi.org/10.1071/RDv32n2Ab20

6. Jeong, Y., Olson, O.P., Lian, C., Lee, E. S., Jeong, Y. W., Hwanga, W. S. (2020). Dog cloning from post-mortem tissue frozen without cryoprotectant. Retrieved from: https://doi.org/10.1016/j.cryobiol.2020.03.013

7. Qu, P., Wang, Y., Zhang, C. & Liu, E. (2020). Insights into the roles of sperm in animal cloning. Stem Cell Res Ther 11, 65. https://doi.org/10.1186/s13287-020-01599-6

8. Zhang, L., Yu, M., Xu, H., Wei, X., Liu, Y., Huang, C., Chen, H., Guo, H. (2020). RNA sequencing revealed the abnormal transcriptional profile in cloned bovine embryos. Retrieved From https://doi.org/10.1016/j.ijbiomac.2020.02.026

9.Carter, W. (2020). Dead as a Dodo? Biochemical Society. Policy. 50-51

10.Shapiro, B. Mammoth 2.0: will genome engineering resurrect extinct species?. Genome Biol 16, 228 (2015). https://doi.org/10.1186/s13059-015-0800-4


Images

DNA-Public Domain Pictures

IVF- DrKontogianniIVF

Cryopreservation-USDA Gene Bank

©2020 by Candid Science Directive.