  Cloning   從複製羊到複製人

1.  Cloning 從複製羊到複製人 Yu-Hui Tsai Ph.D. 台北醫學大學 醫學科學研究所 蔡郁惠

2. Introduction

3. Cell Differentiation(細胞分化) • In the field of biological sciences, there is a dogma that the expression state of genomein differentiated cells during the course of development, especially in the terminal differentiated cells, is irreversible.

4. Cloning Plants from a Single Cell • In plants, the ability of a differentiated cell to develop into a whole new individual is common. A single cell removed from carrot root and placed in culture medium may begin dividing and eventually grow into an adult plant. • In this way, it is possible to propagate/clone large number of plants having desirable traits. • Based on this data, it is evident that differentiation does not necessarily cause irreversible changes in genomic DNA.

6. What is Cloning ? Scientific American (2002): p.18-19

7. What is Cloning ? • Cloning is a (new) biotechnological process involving injecting a diploid nucleus into an unfertilized, enucleated egg and allow it to develop up to blastocytes or a live new born. • Cloning may be subdivided into : • Therapeutic cloning : to produce stem cells for regenerative medicine • Reproductive cloning : to create a body

8. Frog • In 1952, American embryologists Robert Briggs and Thomas King performed nuclear-transfer experiments with frog. They found that the number of embryos surviving to become tadpoles was smaller when donor cells were taken from animals at a more advanced developmental stage. Only 1 to 2 percent of embryos survived to become live offspring. Some of those clones that survive through birth die shortly afterward.

10. Cloning for medicine Scientific American (1998): p.58-63

11. Megan and Morage :clones of embryonic cells • In 1995, Scientists at Roslin Institute near Edinburgh, Scotland, managed toproduce Megan and Morage from cultured cells derived from a nine-day-old embryo. This made Megan and Morage clones of the embryo. • Thus, partially differentiated cells can be genetically reprogrammed to function like those in an early embryo.

14. Clones from cells of a matured animal • In February 1997, Dolly was the first mammal cloned from a cell of matured ewe’s. • Genetic tests prove that Dolly is a clone of fully differentiated mammary cell of an adult. • All cloned offspring look like the breed of sheep that donated nucleus.

17. Dolly give birth to Bonnie, which is normal, in 1998. Later, Bonnie delivered a healthy set of triplet.

18. Clone with a Genetically Modified Cell • The advancement in cell biotechnology, especially genetic recombination, makes it easy to make genetically modified animals. • A genetically modified cell may be used as donor for nuclear transfer, the resulting cloned offsprings will all carry the construct. • The first transgenic sheep was born in 1997 (Summer), Polly and others secrete the human protein in their milk.

21. Cloning Examples in Taiwan • 一個研究團隊於91年2月15日育成帶有人類第九凝血因子及豬乳鐵蛋白轉殖的複製豬，是全世界首例。 • 畜試所於91年7月5日、以體細胞複製的雙胞胎複製羊誕生，活存良好，亦為世界首例。

22. Oocyte.

23. Cloned calves • Scientists at Advanced Cell Technology reported on November 30, 2001 (Science) that 26 out of 30 birth of cloned cattle survived. • Telomeres from cloned calves are just as long as those from normal calves. • It appears that the cloning process reset the “aging clock” in cloned cells, which appear younger in some ways than the original cells donated nuclei.

24. The First Human Cloned Embryo Scientific American (1997): p.122-123

25. In 2001, scientists at Advanced Cell Technology in Worcester, Mass. USA successfully cloned the first human embryos using the technique of nuclear transplantation.

26. How to Clone • Cloning is based on nuclear transfer • It usually involves the use of two cells. The recipient cell is usually an unfertilized egg soon after ovulation. • The donor cell, complete with its nucleus whose DNA is not duplicated, fused with the recipient egg begins to develop like a normal embryo and produces offspring if implanted into the uterus of a surrogate mother.

29. Problems comes with cloning human • Ethic problem: who, intention, legality • Human right, identity • Social problem and Legality

30. Nature 436 (2005), 641-642

32. Other animals cloned • Rhesus Monkey: Tetra (female, January 2000) from embryo splitting • Cattle: Alpha and Beta (males, 2001) and (2005) Brazil • Cat: December 2001, scientists at Texas A&M University, the first cat cloned. CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons • Mule: Idaho Gem (male, 2003-05-04), was the first horse-family clone. Utah Pioneer (male, June 2003) Idaho Star (male, July 2003) • Rabbit: In France and North Korea independently (March-April, 2003) • Deer: Dewey (2003) • Dog:Japanese Scientists have cloned a Great Dane named Scooby Doo. • Horse: Prometea, a Haflinger female born 2003-05-28, was the first horse clone; Paris Texas (male, March 2005)

33. What Cloning Means for Gene Therapy? Scientific American (1997): p.122-123

35. Parthenogenesis • Cloned early-stage human embryos generated only from eggs prior to meiosis II. Stem cells derived from such Parthenon-genetically activated cells would be unlikely to be rejected after transplantation to the egg donor herself. (However, the stem cells from such embryo would not be genetically identical to the donor’s.)

36. Androgenesis • A process involves transferring two nuclei from the man’s sperms into a contributed egg that had been stripped of its nucleus in an attempt to create stem cells for treating the man’s disorders.

37. What possibilities can cloning offer ? 1. Cloning of modified porcine/sheep mammary cells containing various human gene constructs to generate animals which will secret various desirable proteins in the milk. 2. Creating large animals carrying genetic defects that mimic human genetic disorders that are good models for evaluating their long-term responses to treatments.

38. What possibilities can cloning offer ? 3. Generating genetically modified animal organs that are suitable for transplantation into human. 4. Creating animals with a precisely engineered genetic constitution that can be employed in cell based therapies for illness such as parkinson’s disease, diabetes, muscular dystrophy. 5. Producing herds of cattle free of prion protein gene.

39. What possibilities can cloning offer ? 6. Creating universal human donor cells for medical usage. 7. Establishing permanent, stable human embryonic stem-cell lines for regenerative medicine and gene therapy. 8. Ideally, manufacturing genetically matched stem cells for a patient by “dedifferentiating” them directly without utilizing an embryo is a much superior strategy.

43. Other References • www.sciam.com/explorations/2001/11240/ezzell. • http://www.sciscape.org/news_detail.php?news_id=592 (複製豬─異種器官移植的明日之星, 2002);Lai, L. et al. Production of α-1,3- Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning. Science 2002 Jan 3. • www.c-science.com/txt/tc/sc/ls/000819scls.htm - 5k (Cloned pigs may offer organs for transplant, 2000) • http://e-info.org.tw/node/19769 (複製豬：為延續人類生命或滿足過度消費 2007)

44. The End Thank you !

45. Chimeric embryo