Animal Cloning

1. Animal Cloning ES cells

2. Embryonic stem (ES) cells Pluripotent stem cells derived from the inner cell mass of the blastocyst Can be cultured, manipulated and then reinjected into blastocysts, where they can go on to contribute to all parts of embryo. In principle, ES cells also might be able to generate large quantities of any desired cell for transplantation into patients.

3. Human embryonic stem cells (hES cells) • hES cells represent the earliest stage of cells in human development. • hES cells may differentiate into any one of the 230 different cells in human body. • hES cells may treat many human diseases.

4. hES cell development stages

5. 治疗多种疾病 Treat many diseases 人的胚胎干细胞 Human Embryonic Cells 胚状体细胞 Embryoid Bodies Dopamine 神经原 Dopamine neurons 震颤性麻痹 Parkingson’s disease 树状神经原 Oligodendrocytes 脊髓损伤 Spinal cord injury 心肌细胞 Cardiomyocytes 心脏病 Heart disease 胰岛细胞 Islet beta cells 糖尿病 Diabetes 肝脏细胞 Hepatocytes 肝脏疾病 Liver diseases 成骨细胞 Osteoblasts 骨质疏松症 Osteoporosis 软骨细胞 Chondrocytes 骨关节炎 Osteoarthritis 其他细胞 Other cells 其他疾病 Other Diseases hES may differentiate into any human cells.

6. Mouse embryonic stem cell cultures - LIF LIF (leukaemia inhibitory factor) maintains embryonic stem cells in an undifferentiated state ES cells spontaneously differentiate when allowed to aggregate in the absence of LIF

7. Human stem cell lines available (August 28, 2001) http://www.the-funneled-web.com/images/Embryonic%20stem%20cells.gif

8. Totipotent and pluripotent cells Totipotent = meaning that its potential is total. isolated directly from the inner cell mass of embryos at the blastocyst stage. (IVF-IT surplus embryos in case of humans) pluripotent = they can give rise to many types of cells but not all types of cells (no fetus developed). www.laskerfoundation.org/ news/weis/estemcell.html

9. Adult stem cells multipotent but not totipotent

10. More about stem cells Embryonic stem cells Adult stem cells Truly pluripotential More restricted pattern of differentiation several countries have sanctioned deriving human ES-cell lines from ‘surplus’ embryos created through in vitro fertilization medical gain without ethical pain

11. ENUCLEATION cytoplast oocyte Nuclear Transplantation Nucleus comes from someone to be cloned 1. Enucleation of the cell 2. Nuclear transfer removal of the nucleus chromosomes are gently sucked out with a sharp micropipette A. electrofusion whole donor cell injected beneath the zona pellucida (the outer membrane of the oocyte) and fusion of cells induced by electrical impulses From the a mature unfertilized oocyte (egg) Or from the cell in quiescent state (inactive G0 phase of cell cycle) OR metaphase II B. nuclear injection naked nucleus microinjected into cytoplast

12. Electrofusion http://www.brinkmann.com/pdf/cell_fusion.pdf fusion pulse Cells brought close together Heterokaryon phase: nuclei distinct fusion product Fusion induced by electric pulse

13. Genetic Reprogramming If cell for cloning taken from adult organism “de-differentiation” – rearranging the genome of the nucleus to restore its totipotency so it can differentiate into different types of cells and develop into a whole organism must occur after nuclear transfer to successfully produce the clone – required for the nuclei from adult cells to develop normally best completed in unfertilized oocytes (as plasma donors)

14. Re-programming never achieved with same success as fertilization Fig. 5 from Nature Reviews Genetics 3: 671

15. Development of the embryos from cell with “alien” nucleus May be induced by chemical treatments Developing embryos are grown in a culture to assess their viability Implantation of Embryo embryos are surgically transferred into the uteri of suitable surrogate mothers many embryos are transferred to each surrogate mothers to ensure implantation

19. Therapeutic Cloning

21. Why human Therateutic cloning is unethical in USA? 􀁗 1. Therapeutic cloning can only be justified by the utilitarian calculus that values potential medical treatments over the lives of the embryos who would be destroyed in order for the research to proceed. However it is not ethical to sacrifice one human life for the real or potential benefit of others. 􀁗 2. It is unethical to view a human being - regardless of its age-as a means to an end. Creation of human embryos specifically for destructive research is opposed by our community, and this is what is involved in therapeutic cloning.

22. 􀁗 3. Therapeutic cloning will undoubtedly lead to exploitation of women. In order to create human clones for stem cell therapy, an enormous number of women's eggs will need to be donated. To do so, women may be treated with superovulatory drugs and must undergo an invasive procedure. Complications may occur. Advanced cell technology paid up to $4,000 to each woman who donated eggs for their cloning experiments. It is likely that women of lower economic status will be exploited in this way. 􀁗 4. In addition to the ethical concerns above, therapeutic cloning should be banned because it increases the likelihood of reproductive cloning. Preventing the implanting and subsequent birth of cloned embryos once they are available in the laboratory will be impossible. Already Dr Severino Antinori of Italy has plans to produce the first cloned human. The most effective way to prevent reproductive cloning is to stop the process at the beginning, with the creation of cloned embryos. Since the overwhelming community consensus is that reproductive cloning should be banned, steps must be taken to ban therapeutic cloning as well.

23. 􀁗5. Embryonic stem cell research and therapeutic cloning should also be banned in view of the existence of other promising and ethical treatment options such as adult stem cell therapy (which has already been successfully used in patients). 􀁗6. In conclusion, if destruction of excess embryos is allowed, does this just apply to the embryos currently in storage, or to future 'excess' embryos as well? The blatant irresponsibility of IVF clinics that have allowed the accumulation of approximately 70,000 embryos to date will have no incentive to change if future excess embryos are also fodder for the laboratory.

24. 1984 – A live lamb was cloned from sheep embryo cells Mammal Cloning Timeline 1986 – Early embryo cells were used to clone a cow 1993 – Calves were produced by transfer of nuclei from cultured embryonic cells 1995 – Two sheep, named Megan & Morag, were cloned using embryo cells 1996 – Birth of Dolly, the first organism to be cloned from a fully differentiated adult cell 1997 – Transgenic sheep named Polly was cloned containing a human gene Megan and Morag Dolly http://www.cnn.com/2001/WORLD/europe/08/06/clone.critics/index.html

25. 1998 – 50 mice were cloned in three generations from a single mouse Tetra 1998 – 8 calves were cloned from a single adult cow, but only 4 survived to their first birthday 1999 – A female rhesus monkey named Tetra was cloned by splitting early embryo cells. 2000 – Pigs and goats reported cloned from adult cells 2002 – Rabbits and a kitten reported cloned from adult cells http://hs.houstonisd.org/hspva/academic/Science/Thinkquest/gail/text/benefits.html

26. Dolly with her surrogate mother Dolly • Born in July 1996 at the Roslin Institute in Scotland • First mammal to be cloned from an adult mammal using the nuclear transfer technique • 277 attempts were made before the experiment was successful • Dolly died in February 14, 2003 of progressive lung disease at the age of 6; whereas normal sheep can live up to 12 years of age. Dolly with her first newborn, Bonnie

27. Mammal Cloning allows propagation of endangered species http://www.howstuffworks.com/cloning.htm/printable January 8, 2001 Noah, a baby bull gaur, became the first clone of an endangered animal.

28. Species Number of oocytes used Number of live offspring Notes Mouse 2468 31 (1.3%) - Bovine 440 6 (1.4%) 2 died Sheep 417 14 (3.4%) 11 died within 6 months Pig 977 5 (0.5%) - Goat 285 3 (1.1%) - Comparison of Cloning Success Rates in Various Animals The table shows success rates of cloning when mature mammal cells were used. Yanagimachi, R.  2002.  "Cloning: experience from the mouse and other animals." Molecular and Cellular Endocrinology.  21 March, 187.

29. Development and survival of cloned mouse embryos Majority of the embryos die before and after implantation. This figure shows that the present cloning technique is highly inefficient. Yanagimachi, R.  2002.  "Cloning: experience from the mouse and other animals." Molecular and Cellular Endocrinology.  21 March, 187.

30. Clone Birth Defects • Cloned offspring often suffer from large offspring syndrome, where the clone and the placenta that nourished it are unusually large. • Cloned offspring often have serious inexplicable respiratory or circulatory problems, which causes them to die soon after birth. • Clones tend to have weakened immune systems and sometimes suffer from total immune system failure. • Very few clones actually survive to adulthood. Clones appear to age faster than normal. Clones experience problems associated with old age, such as arthritis, while they are still young. This may be due to the fact that clones have shorter telomeres

31. The whole story about cloning is not a reproductive story The possibility of using cloning technology to grow organs genetically identical to our own for transplantation – thereby avoiding rejection of foreign issues http://medlib.med.utah.edu/WebPath/CVHTML/CV001.html

32. http://easyweb.easynet.co.uk/~sfl/rlb3a.jpg