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--- group_2_presentation_2_-_cloning [2020/02/26 22:37]
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 ====== What is Cloning? ======
-Clones are referred to as organisms that are exact genetic copies of each other. To say the least, every single piece of their DNA sequence is identical ("Cloning LiteratureWatch '', 2000). Till now, researchers have cloned various biological organisms from single cells and tissues to whole organisms such as a sheep (Noh & Neumann, 2001). Cloning is a process that can occur both naturally and artificially in the lab through the help of scientists (Noh & Neumann, 2001). Natural cloning is simply known as asexual reproduction in which plants and other single-celled microrganisms are able to produce their genetically identical counterparts readily (Wilmut, 2002). However, when it comes to artificial cloning, technological advances have paved way for newer techniques to arise (Noh & Neumann, 2001). According to the National Human Genome Research Institute (NHGRI), these include gene cloning, reproductive cloning and therapeutic cloning (Noh & Neumann, 2001).
+Clones are referred to as organisms that are exact genetic copies of each other. To say the least, every single piece of their DNA sequence is identical ("Cloning LiteratureWatch '', 2000). Cloning can occur both naturally and artificially. We observe many examples of natural cloning around us, for example bacteria reproducing asexually through the process of binary fission is an example of cloning (Noh & Neumann, 2001). Our cells undergo cloning everyday through mitosis. And lastly, another really cool example of cloning are identical twins. All these events have inspired scientists to experiment cloning through artificial means. Science has gone from cloning single cells to entire organisms and advancements continue to happen today (Noh & Neumann, 2001). The three types of artificial cloning that we will be discussing today include reproductive cloning, therapeutic cloning and gene cloning (Noh & Neumann, 2001).
-Gene cloning initiates with the extraction of a DNA molecule from an organism and breaking the individual nucleotides down by slicing the strand into small pieces (Wilmut & Dominko, 2000). Plasmids are then integrated into genes and subsequently transferred into living bacteria and subsequently made into millions of colonies (Wilmut & Dominko, 2000). After pinpointing a particular gene of interest, bacteria is propagated to make multiple copies for further analysis (Wilmut & Dominko, 2000). These copies can then be utilized for modifying certain gene sequences in which scientists have the option of integrating novel pieces of DNA and replace it with existing segments of genomic material (Wilmut & Dominko, 2000).
-On the other hand, in reproductive cloning, scientist removes a mature cell, usually a somatic cell, from an organism and transfers it to an egg that recently had its own DNA taken out (Seidel, 2004). That same egg is then implanted into the uterus of a female with the same species of the egg (Seidel, 2004). This was the exact technique used in forming the well known “Dolly the Sheep” (Seidel, 2004).
-Lastly, in therapeutic cloning, a sample cell, similar to reproductive cloning, is taken out from an organism and is transferred to an outer membrane of a donor egg cell ("Cloning LiteratureWatch", 2000). This allows the egg to create embryonic stem cells ("Cloning LiteratureWatch", 2000).
+<box 50% round center|> {{:identical.png?500|}} </box| Figure 1. Clone humans with identical Dna sequences. (Parsons, 2008)>
 ====== History ======
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 ==== Reproductive Cloning of organisms ====
-It is a method of creating clones using scientific technology. There are two ways of creating clones artificially, each with their own advantages and drawbacks.
+The two methods of artificially creating clones include Embryonic Splitting and Somatic Cell Nuclear Transfer (SCNT).
-Embryonic splitting.
+==Embryonic splitting==
-This technique is inspired by the formation of identical twins in organisms. This was one of the initial approaches towards creating clones artificially (Keefer, 2015). Embryonic splitting can be further broken down into two different procedures called the blastomere splitting for cleavage stage, or bisection if the fertilized egg has reached the blastocyst or morula stage. Embryonic splitting is a simpler method of creating clones, however it results in only a limited number of clones. Performing embryonic splitting at later stages may cause non-viable embryos due to a loss of mass. This is due to the fact that the initial stages of division following fertilization involve cell division without any significant overall cell growth.
+This technique is inspired by the formation of identical twins in organisms. This was one of the initial approaches towards creating clones artificially (Keefer, 2015). Embryonic splitting can be further broken down into two different procedures called the blastomere splitting for cleavage stage, or bisection if the fertilized egg has reached the blastocyst or morula stage. Embryonic splitting is a simpler method of creating clones, however it results in only a limited number of clones. The limitation is due fact that the initial stages of division following fertilization involve cell division without any significant overall cell growth. Thus, too much splitting can lead to a loss of mass necessary to give rise to a viable organism (Keefer, 2015).
 Blastomere splitting involves the separation of blastomeres from an early embryo to form two or more embryos. The process involves removal of one or more blastomeres from a cleavage stage embryo followed by insertion into an empty Zona Pellucida (the protective membrane of an oocyte). Prior to insertion through the Zona Pellucida, the donor blastomere is treated with acidified tyrode’s solution to make an opening in the donor cell Zona Pellucida. Then using an aspirating pipette the blastomere is removed and then inserted into a separate empty Zona Pellucida (Noli, Ogilvie, Khalaf and  Ilic, 2017).
-             Figure 1: Blastomere splitting to create one or more embryos.
+<box 50% round center|> {{:blasto.png?500|}} </box| Figure 2. Blastomere splitting to create one or more embryos. (Noli, Ogilvie, Khalaf and  Ilic, 2017)>
-Another method of performing embryonic splitting is by performing a bisection on post-compacted embryo (stage following cleavage when the egg consists of the inner cellular mass (ICM) and trophoblast). Bisection involves precisely cutting the embryo and thus the ICM in half using a surgical microblade (Noli et al, 2017).
+Another method of performing embryonic splitting is by performing a bisection on post-compacted embryo. An embryo in the post-compacted stage is differentiated into an inner cellular mass (ICM) and the trophoblast which eventually becomes the placenta. . Bisection involves precisely cutting the embryo and thus the ICM in half using a surgical microblade (Noli et al, 2017).
-Figure 2: Bisection of a post-compacted embryo to achieve embryonic splitting.
+<box 60% round center|> {{:bisection.png?500|}} </box| Figure 3: Bisection of a post-compacted embryo to achieve embryonic splitting. (Noli et al, 2017)>
 ==== Somatic Cell Nuclear Transfer (SCNT) ====
-Compared to embryonic splitting, theoretically this method should be capable of producing multiple clones. The first stage of SCNT involves preparation of the somatic cell, typically a skin fibroblast as it is easy to access and is less invasive and painful (Wilmut, Schnieke, McWhir, Kind and Campbell, 1997). To retrieve the cells, a tiny amount of skin is obtained and placed in a trypsin enzyme buffer solution that allows the cell to detach from its surrounding extracellular matrix. The cell is then placed in a serum medium for three weeks to obtain a single layer containing only fibroblasts.  The second stage involves the preparation of an oocyte. To do this, Human Chorionic Gonadotropin Hormone (hCG) is used to induce ovulation (French et al, 2008). The ovulated egg is extracted and the egg’s DNA is fluorescently labelled to assist with efficient removal (Heindryckx, De Sutter, Gerris, Dhont and Van der Elst, 2007). Once the nucleus is removed from the oocyte, it’s referred to as a cytoplast. The karyoplast or the nucleus of the somatic cell is transferred into the cytoplast. Electrical pulses are then used to induce fusion between the karyoplast and the cytoplast to form an activated oocyte. The oocyte contains factors or chemicals that allow it to reprogram the genetic information in the somatic nucleus to induce pluripotency. The oocyte is then implanted in a surrogate mother who carries the pregnancy until delivery (Niemann and Lucas‐Hahn, 2012).
+Compared to embryonic splitting, theoretically this method should be capable of producing multiple clones as it does not involve any loss of mass, however further improvements are required to reach a significant success rate. The first stage of SCNT involves preparation of the somatic cell, typically a skin fibroblast as it is easy to access and is less invasive and painful (Wilmut, Schnieke, McWhir, Kind and Campbell, 1997). To retrieve the cells, a tiny amount of skin is obtained and placed in a trypsin enzyme buffer solution that allows the cell to detach from its surrounding extracellular matrix. The cell is then placed in a serum medium for three weeks to obtain a single layer containing only fibroblasts. Once the skin fibroblasts are cultured, the nucleus or karyoplast is isolated to be later inserted in an egg without a nucleus.The second stage involves the preparation of an oocyte. To do this, Human Chorionic Gonadotropin Hormone (hCG) is used to induce ovulation (French et al, 2008). The ovulated egg is extracted and the egg’s DNA is fluorescently labelled to assist with efficient removal (Heindryckx, De Sutter, Gerris, Dhont and Van der Elst, 2007). Once the nucleus is removed from the oocyte, it’s referred to as a cytoplast. The karyoplast obtained from the skin fibroblasts is transferred into the cytoplast. Electrical pulses are then used to induce fusion between the karyoplast and the cytoplast which results in an activated oocyte. The oocyte contains factors or chemicals that allow it to reprogram the genetic information in the somatic nucleus to induce pluripotency. The oocyte is then implanted in a surrogate mother who carries the pregnancy until delivery (Niemann & Lucas‐Hahn, 2012).
-(Keefer, 2015)
+<box 50% round center|> {{:scnt.jpg?500|}} </box| Figure 4: Artificial cloning of domestic animals carried out thru a process known as Somatic Cell Nuclear Transfer (SCNT). (Keefer, 2015)>
  ==== Human Cloning ====
-Several claims have been made in the past, but all without evidence. Human cloning is scientifically a more complicated and sensitive process, with many ethical concerns attached to it. It is also currently banned in many countries (“Cloning Fact Sheet”, 2017). Scientifically speaking, it is quite challenging to ensure that the human clones created would lead a normal, healthy life. Clones of other organisms created in the past have faced a lot of health challenges prenatally and postnatally and many die at a young age. Thus, similar concerns regarding human cloning pose an ethical challenge.
+Several claims have been made in the past, but all without evidence. Human cloning is scientifically a more complicated and sensitive process, with many ethical concerns attached to it. It is also currently banned in many countries (“Cloning Fact Sheet”, 2017). Scientifically speaking, it is quite challenging to ensure that the human clones created would lead a normal, healthy life. Clones of other organisms created in the past have faced a lot of health challenges pre-natally and post-natally and many die at a young age. Thus, similar concerns regarding human cloning pose an ethical challenge.
 ====== Current advancements and Lifespan ======
-There have been 23 species that have been cloned thus far including pigs, goats, rabbits, sheep, cattle and even two rhesus monkeys (“Cloning Fact Sheet”, 2017). The lifespan of these species varies and many are afflicted with abnormalities leading to either abortion or an early death after birth (Burgstaller & Brem, 2017). These abnormalities include increased birth size, organ defects, premature aging and issues in the immune system. Despite these anomalies, certain clones of goats, and mice have been able to survive the maximum lifespan for their species. Some argue that the abnormalities observed could be due to factors like telomere aging and accumulated DNA damage in the donor DNA, however, it has also been observed that oocyte reprogramming is capable of restoring telomere length and other abnormalities (Burgstaller & Brem, 2017). First cloned mammal Dolly survived for 6 years, and died due to a respiratory illness. Currently, the longest living cloned organism is a goat in China called Yangyang cloned in 2000 and according to recent news is still alive.
+Over 20 species of mammals have been cloned thus far including pigs, goats, rabbits, sheep, cattle and even two rhesus monkeys  (Greshke, 2018). The lifespan of these species varies and many are afflicted with abnormalities leading to either abortion or an early death after birth (Burgstaller & Brem, 2017). These abnormalities include increased birth size, organ defects, premature aging and issues in the immune system. Despite these anomalies, certain clones of goats, and mice have been able to survive the maximum lifespan for their species. Some argue that the abnormalities observed could be due to factors like telomere aging and accumulated DNA damage in the donor DNA, however, it has also been observed that oocyte reprogramming is capable of restoring telomere length and other abnormalities (Burgstaller & Brem, 2017). Hence, it could be speculated that the probability of cloning a healthy organism depends on the ability of these occyte factors to fully reprogram the genetic information. Despite
+these anomalies, certain clones of goats, and mice have been able to survive the maximum lifespan for their species. Currently, the longest living cloned organism is a goat in China called Yangyang cloned in 2000 and according to recent news is still alive (Burgstaller & Brem, 2017).
-Transgenic organisms can also be cloned via somatic cell nuclear transfer. Organisms containing foreign DNA, that makes their traits desirable can be cloned using the SCNT method. The Scottish researchers responsible for cloning Dolly the sheep, also genetically modified and cloned other sheep to produce a human clotting factor protein, bio-thrombin in its milk.
+Transgenic organisms can also be cloned via somatic cell nuclear transfer. Organisms containing foreign DNA, that makes their traits desirable can be cloned using the SCNT method. The Scottish researchers responsible for cloning Dolly the sheep, also genetically modified and cloned other sheep to produce a human clotting factor protein, bio-thrombin in its milk in the hopes of providing treatment for individuals lacking the protein. Other experiments have been performed with transgenic clones containing genes for industrial proteins as well as human polyclonal antibodies ("Cloning Fact Sheet", 2017).
 Furthermore, cloned animals have also been used for clinical trials, allowing scientists to observe uniform responses for a given genetic makeup since all model organisms are genetically identical. In 2008, FDA approved meat and milk from cloned animals as safe to consume, which allows scientists to freely clone organisms with the most desirable traits, however, due to the cost associated with cloning it will still take some time for these products to become available to the public (“Cloning Fact Sheet”, 2017).
-====== Advanced cloning ======
+<box 50% round center|> {{:collection.png?600|}} </box| Figure 5: The number of animals that have been cloned so far: (A)Rhesus Monkey (Sartore, n.d.), (B) Pigs (“Cloned pigs”, 2000), (C) Cat (Wadsworth,n.d.), (D) Horse (Pabst, 2017), (E) Rabbit (“Cloned Rabbit, n.d.) >
+====== Other Types of Artificial Cloning ======
 Reproductive cloning, therapeutic cloning and gene cloning are three types of artificial cloning. Reproductive cloning produces an animal that is genetically identical to the donor animal (Cloning Fact Sheet, 2017). This is done through somatic cell nuclear transfer (SCNT). Reproductive cloning involves implantation of a cloned embryo into a uterus (Rugnetta, 2019). This allows the embryo to develop into a fetus. This type of cloning experiment was performed for over 40 years through embryo splitting (Rugnetta, 2019). In the 1990s, reproductive cloning saw major changes. This was after the birth of “Dolly the sheep”. Dolly was generated through SCNT (Rugnetta, 2019). Reproductive cloning using SCNT is considered to be very harmful because fetuses of embryos cloned through this process are usually born with birth defects. Also, fetuses born this way rarely survive gestation (Rugnetta, 2019).
@@ Line 70: / Line 69: @@
 Although much research is still being conducted in regards to cloning extinct and endangered species, many attempts have been done in the past by researchers (White, 2000). One of the first exposures was in 2003 when the first extinct-animal clone was created (White, 2000). How was it exactly created? A sample of frozen skin was extracted to clone a bucardo, also known as Pyrenean ibex (White, 2000). This was a subspecies of the spanish ibex that went extinct originally in 2000 (White, 2000). Even though it was one of the successful attempts, the bucardo died shortly after birth (White, 2000). However, upon examination, the process was tedious to say the least. The scientists first cloned the embryos by inserting the bucardo's DNA into domestic goat eggs removed from their original genetic material ("Cloning LiteratureWatch", 2000). Initially, researchers implanted about 208 embryos, of which only 7 goats became pregnant, and 1 survived ("Cloning LiteratureWatch", 2000). Through further investigation, researchers noted that the ibex eventually died from respiratory failure due to significant lung abnormalities right after its birth ("Cloning LiteratureWatch", 2000).
+<box 40% round center|> {{:bucardo.png?350|}} </box| Figure 6: The First Extinct Mammal Brought Back to Life—Briefly—Was the Bucardo . Retrieved from https://www.jstor.org/stable/90006702?seq=2#metadata_info_tab_contents>
 Other researchers soon followed and cloned two primates using the same technique that gave rise to Dolly the sheep (Wilmut, 1999). Being the first time in history in which primates have been cloned in an effective and appropriate manner, this outlined a major achievement in cloning history (Wilmut, 1999). After years-long experimentation, the chinese researchers named the two female macaques Zhong Zhong and Hua Hua, are genetically identical and were born after a years-long effort (Wilmut, 1999). This discovery along with other milestones gave hope and for replicating the same technique in endangered species (Noh & Neumann, 2001).
@@ Line 90: / Line 91: @@
-Retrieved from: https://bioethicsarchive.georgetown.edu/pcbe/reports/cloningreport/fullreport.html
+<box 40% round center|> {{:diary.png?300|}} </box| Figure 7: The process of therapeutic cloning and derivation of pluripotent stem cells. Retrieved from: https://bioethicsarchive.georgetown.edu/pcbe/reports/cloningreport/fullreport.html>
 One of the biggest concern comes from the basic fact that cloning would allow humans to procreate in an asexual manner which would give us the capacity for complete control over the genetic profile of our children. Upon further investigation fears about harm to the children's psychology and personality were revealed (Shapiro, 1997). Majority of these fears arise from opponents and their concerns that the technology is not yet safe enough and could be abused (AAAS Scientific Responsibility, Human Rights and Law Program, 2002). In addition to being harmful to the child itself, some expressed a concern for its negative effects on important social values. Social concerns such as how these cloned individuals will be able to integrate themselves within their societies and families (McGee, 2000).  As this somatic cell nuclear transfer cloning would open doors to a form of eugenics or tempting some to manipulate others as if they were objects instead of people with emotions and lives (Sapiro, 1997). Religion also played a role in the ethics of human cloning. Many religious individuals are against the technology because it is taking ‘God’s Place’ and because this will be leading to destroying a human life (in this case an embryo) (Sullivan, 2003). However, there are a few religious groups who support therapeutic cloning because of its potential life-saving benefits (Bainbridge, 2003).
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-Figure X:
+<box 80% round center|> {{:cumulus.png?1000|}} </box| Figure 8: The process of therapeutic cloning and derivation of pluripotent stem cells. (Cibelli et al, 2001)>
+Link to powerpoint presentation: https://1drv.ms/p/s!AkW_XMx1tFYxumquXn_Knr16kTFC?e=fGa3br

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