Dr. B. Victor is a retired biology professor with over 32 years of experience teaching and researching reproductive technology in fishes. His presentation outlines various forms of reproduction including asexual, sexual, and parthenogenesis. It also discusses cloning technology such as embryo splitting, nuclear transfer, and the three main types of cloning - recombinant DNA cloning, reproductive cloning, and therapeutic cloning. The benefits and applications of cloning as well as techniques for transgenic animal production are also summarized.

1. Reproductive Biotechnology Dr. B. Victor, St. Xavier’s College, Tirunelveli-2

2. About the presenter Dr.B.Victor is a highly experienced postgraduate biology teacher, recently retired from the reputed educational institution St. Xavier’ s College, Palayamkottai, India-627001. He was the dean of sciences and assistant controller of examinations. He has more than 32 years of teaching and research experience His research interest revolve around reproductive technology of Fishes. Send your comments to : bonfiliusvictor@gmail.com

3. Presentation outline Kinds of reproduction Annual Breeding Cycles Reproduction and Development Cloning Technology Types Of Cloning recombinant DNA cloning, therapeutic cloning reproductive cloning Embryo splitting, Nuclear Transfer Technology Benefits of human cloning

4. Kinds of reproduction Asexual reproduction Inferior form of reproduction. Requires one parent animal. No involvement of gametes. Offspring identical to their parent. Mitotic chromosome division (Binary fission) E.g., protozoans, coelenterates

5. Cloning / Asexual reproduction

6. Sexual reproduction . Requires two parents. Involvement of gametes. Meiotic chromosome division. Fusion of gametes results in fertilization Reshuffling of parental genes. Offspring not identical to their parents

7. Sexual reproduction

8. Parthenogenesis Development of an egg without fertilization by sperm. Offspring genetically identical to their parents. Invertebrates ( e.g., arthropods) Vertebrates ( e.g., some fish )

9. Parthenogenesis

10. Reproductive Cycles Annual Breeding Cycles Continuous breeders Breeds throughout the year e.g., human,. guinea pig, rat. Seasonal breeders Breeds during specific seasons e.g., dog , cat, sheep. cattle Oestrus cycle Females sexually receptive during heat period e.g., cow, rat, pig Menstrual cycle Periodical discharge of menstruation e.g., monkeys. Ape, man

11. Reproduction and Development

12. Human Ovary

13. Reproductive hormonal cycles

14. Oogenesis

15. Oogenesis

16. Seminiferous tubule

17. Sperm

18. Human development

19. Human- Early development

20. Human blastocyst

21. Human Life cycle

22. Cloning Technology

23. What Is Cloning? Cloning is the creation of an exact genetic replica of a small segment of DNA, a cell or a whole organism.

24. Natural cloning : Identical twins and multiple births are an example of human clones that are created naturally. Artificial cloning : Dolly, the cloned sheep, was created artificially in a laboratory in Scotland in 1997 (Nature, 385, 810- 13). Types of Cloning

25. Natural Cloning Cloning is a form of asexual reproduction which is widespread in nature. In single-cell organisms and plants, it is an entirely normal process (division, vegetative reproduction),

26. Animal Cloning Animals can be cloned by embryo splitting or nuclear transfer. Embryo splitting involves bisecting the multicellular embryo at an early stage of development to generate "twins". This type of cloning occurs naturally and has also been performed in the laboratory with a number of animal species.

27. History of Cloning: 1952 – Robert Briggs and Thomas King of the Institute for Cancer Research, developed the first major technique, Somatic Cell Nuclear Transfer (SCNT) 1966 – John Gurdon of Oxford University created an adult frog clone using a tadpole somatic cell. 1980 – Embryo splitting, developed for livestock breeding. 1980 – 1996 –Various research groups cloned frogs, mice, and cattle. 1996 –Ian Wilmut and colleagues of Roslin Institute in Scotland created the first clone of an adult mammal using adult somatic cells and SCNT. Dolly, the famous cloned sheep is born.

28. History of Cloning: 1997 – Ian Wilmut and colleagues cloned two other sheep, Molly and Polly. 1998 –Univ. of Hawaii announced the creation of 50 mice clones using adult cells. 2000 – PPL Therapeutics, Inc. cloned pigs from adult female pig body cells. 2001 – PPL Therapeutics, Inc. applied genetic engineering to pig cloning, so that the pigs contain a jellyfish gene. 2004 – South Korean scientists achieved the first successful human somatic cell nuclear transfer.

29. TYPES OF CLONING 1. DNA Cloning DNA cloning is also known as recombinant DNA technology, molecular cloning and gene cloning. Gene cloning is also important for the development of drugs and treatments such as in pharmacogenetics and gene therapy

30. TYPES OF CLONING 2. . Reproductive Cloning Reproductive cloning is also called adult DNA cloning. The purpose is to produce a genetic duplicate of an existing or previously existing organism.

31. Traditional Reproduction and Cloning

32. Types of Genetic Cloning recombinant DNA cloning, reproductive cloning therapeutic cloning

33. Recombinant DNA Cloning Recombinant DNA cloning or gene cloning refers to the process by which a fragment of DNA is transferred from one organism to a self-replicating genetic element such as a bacterial plasmid or a virus. Plasmids are self-replicating extra-chromosomal circular DNA molecules and can be used to make many copies of the gene. These genetic elements can then be inserted into a host cell of interest and the function of the gene of interest studied.

34. Reproductive Cloning Reproductive cloning refers to the process by which an animal is created which had the same nuclear DNA as a previously existing animal. This is accomplished by the removal of the DNA containing nucleus of an egg and its replacement by the DNA from another cell. The egg is then stimulated to divide and following a number of divisions it can be transferred into the uterus of a suitable female host until its birth.

35. Therapeutic cloning Therapeutic cloning, or embryo cloning refers to the production of human embryos for research purposes. The goal of this is not to create cloned babies but to harvest the stem cells of the embryo that have the potential to develop into almost any cell in the body

36. Artificial cloning procedures Embryo splitting, Nuclear Transfer Technology

37. Embryo splitting Embryo splitting and nucleus transfer differ in the degree of genetic identity achieved in the resulting embryos. Embryo splitting changes neither the age nor the (toti-)potency of the cells used. The (two) embryos from the splitting are in the same stage of development, exactly the same age as the undivided embryo would have been and genetically completely identical.

38. The nucleus transfer technique Transfer of the genetic program (the cell nucleus with the desired genetic material) from a totipotent blastomere to an unfertilised egg cell whose nucleus has previously been removed. This technique basically offers the possibility of replicating an adult individual and their genetic program.

39. Errors in nuclear-cloning technology First, most clones die early in gestation, and only a few survive to birth or beyond. Second, cloned animals have common abnormalities regardless of the type of donor cell or the species used, and third, these abnormalities correlate with aberrant gene expression, which most likely results from faulty genomic reprogramming. Fourth, the efficiency of cloning depends on the state of differentiation of the donor cell.

40. Applications of DNA cloning 1. Genetic components (whole genes or parts of genes) can be reconstructed into unique combinations not easily achieved by natural selection, or synthetic genes can be constructed- -Recombinant DNA. 2. Recombinant DNA can be introduced or reintroduced into bacteria, plants, or animals. 3. If an organism integrates recombinant DNA into their genome or genetic make-up, they are transgenic.

41. Transgenic Technology An animal whose genetic composition has been altered to include selected genes from other animals or species by methods other than those used in traditional breeding

43. Methods of Producing Transgenics A. Nuclear Transfer/Cloning a. Example: Dolly the Sheep b. Transfect primary cells c. Tranfer nucleus to oocyte B. Gene Targeting a. Infusion of mammary gland with genetically manipulated virus --Similar to retroviral method b. Mitotically active alveoli incorporate gene (s) C. Spermatogonal Transfer a. Transfect Spermatogonia with recombinant DNA b. Inject into Testis

44. Techniques of Reproduction-Related Biotechnologies A. Artificial insemination B. Birth Control Pill C. Estrus synchronization D. Superovulation E. Embryo transfer F. In Vitro Fertilization G. Sperm & Embryo Sexing H. Embryo Splitting I. Transgenic Animals J. Cloning

45. Steps in producing a transgenic animal. Superovulation of Donor animal a. Pregnant Mare Serum Gonadotropin (PMSG) --Follicle Stimulating Hormone-like --Increase number of developing follicles b. Human Chorionic Gonadotropin (hCG) -- Luteinizing Hormone-like --Causes ovulation

46. Oocyte Recovery Ovulation occurs every 21 days ( Cow, horse) and 16 days (sheep, goat).In well managed domestic cattles, 8 – 10 eggs are superovulated. Antral ( graffian follicles) are collected by laproscopic surgery. Recovered follicles are allowed to grow in vitro. Slaughterhouse ovaries also often used in livestock

47. In vitro fertilization 1. Fertilization outside the mother 2. First done in 1959 A. Accomplished technique in rodents, pigs, cows, and humans 3. Must induce sperm capacitation A. Oviductal fluid in media B. New medias have been developed 4. Keep fertilization area small A. Buffer drop under paraffin oil

48. in vitro Fertilization (IVF) IVF is carried out in microdroplets of culture medium. Each microdroplet contain 10 oocytes and 1 million sperms per ml ( one dose)

49. Insertion of Recombinant DNA into Embryo Calcium phosphate precipitation Microinjection Retroviral infection Particle gun delivery Electrophoration

50. In vitro Oocyte Maturation (IVM) a. Cleavage stage to Morula b. New Technology: PCR --Used to determine if genes is incorporated in blastomere's genome at this stage.

51. Embryo Transfer Technology a. Recipients are estrus synchronized with donor during the previous estrous cycle b. Prostaglandin F2-alpha commonly used--Causes luteolysis c. Surgical and non-surgical means are used in the transfers.

52. Gestation and Parturtion In Normal reproduction an animal produces about 4-5 offsprings in a lifetime IVF technology can produce 50-80 offsprings in a lifetime.

53. DNA from offspring analyzed for presence of transgene . a. DNA isolated from tail, blood, etc. b. Amplified by PCR if needed c. Analyzed by DNA fingerprinting

54. Methods of getting Recombinant DNA into the embryo A. Microinjection of recombinant DNA into pronuclei (male) of 1 cell embryo or zygote, before syngamy occurs. a. Most successful method in farm species b. Recombinant DNA must have promotor and polyadenylation signals

55. Retroviral Vectors a. Infect early cleavage stage embryo b. Can only incorporate DNA < 8 kb c. Dangerous

56. Embryonic Stem (ES) Cells a. May eventually replace microinjection b. Technique i. ES cells grown in tissue culture ii. Clonal cell lines selected iii. ES cells injected into inner cell mass of blastocyst iv. Chimeric offspring have transgene c. Problem i. Few ES cells exist for other mammals ii. Recently pig ES cell line developed and used successfully to produce transgenic pig

57. Sperm and Embryo Sexing 1. Production Applications A. Dairy and Egg production require more females B. Meat production prefer males C. Parents could pick the sex of a child 2. Flow Cytometry A. Most successful methods for sperm sexing in animals B. Separates sperm by density (Y sperm have slightly less DNA) by FACs 3. PCR amplification of sex-specific genes now possible for sexing embryos A. PCR = Polymerase Chain Reaction a. Amplifies DNA b. Specific sex-related genes i. HY antigen ii. SRY

58. Nuclear Fusion A. Fusion of same sex gametes (Pronuclei) a. Male/Male—androgenomes b. Female/Female--gynegenomes B. Only develop through blastocyst to gastrula stages a. Female genome needed for conceptus development b. Male genome needed for placental development C. Oocytes can be induced to undergo parthenogenesis a. Electrical shock b. Cold shock c. Hyaluronidase treatment d. Gynegenomes

59. Nuclear Fusion

60. Embryo Splitting 1. Blastomeres must be totipotent --8-16 cell stage 2. Cell Mass cut with glass knife dividing groups of blastomeres 3. One set of cells put into denuded zona pellucida 4. Identical twins result

62. Nuclear Transfer &quot;Cloning&quot; 1. Cell nucleus from another donor put into enucleated recipient A. Resulting offspring is a clone of the donor B. Until &quot;Dolly&quot; all donors were embryonic in origin 2. Frogs done in early 70s 3. Mammals A. Mice (Early 80s) B. Cattle (Mid 80s) C. Sheep (Late 80s)

64. Technique of Nuclear transfer technology First, the donor cells are grown under special conditions in culture. The number of cells can be increased by several orders of magnitude. It is also possible to make genetic modifications and to select just those cells in which the desired modification has occurred and multiply these up. These cells are then fused with an unfertilised egg from which the introduced nucleus can lead to the formation of an embryo. The embryos are then transplanted into sheep and lambs are born naturally. This technology could allow the production of genetically identical groups of animals which possess a desirable genetic trait.

65. Cloning technology of sheep The Egg The unfertilised eggs are flushed out of a sheep which has been induced to produce a larger than normal number of eggs. The Cell Previously a sample of tissue was from the udder of a six year old ewe was taken and cultured in a dish (Dolly 1). The cultured cells are starved to send them into a resting or quiescent state. The fusion A cell is placed beside the egg and an electric current used to fuse the couplet. Culture The reconstructed embryo is put into culture and grows for seven days. Development Embryos which grow successfully are taken and transferred to a sheep which is at the the same stage of the oestrus cycle as the egg. The sheep becomes pregnant and produces a lamb after 21 weeks (Dolly).

67. Applications

68. Animals as drug producers “ Gene pharming&quot;, i.e. the use of transgenic animals to manufacture (human) proteins with therapeutic use, e.g. in their milk. The active ingredients from biogenetic manufacturing processes (such as insulin, blood factors or other human bodily substances) can be obtained in much purer form. Production of active ingredients can be on a large scale and relatively cheap. Hazards to people (pathogens) can be avoided as far as possible by careful testing of drugs.

69. Animal models Cloning could be used in producing transgenic animals as animal models for human diseases. Animal models are used to study the biochemical and physiological processes, human diseases and possible therapies. New drugs can be tested in animal models for their toxicity and pharmacological effect on humans

70. Xenotransplantation Xenotransplantation is transplanting animal organs into humans. Cloning of &quot;donor animals&quot;,are made with the desired genetic modifications using nucleus transfer. the alien animal organ will perform its function in the human recipient.

71. Transgenic clones Animal cloning will increase the genetic knowledge of productive animals. Animal cloning technologies will make the &quot;production&quot; of transgenic animals with modified (agricultural) characteristics. The goals for gene transfer in livestock breeding in combination with cloning are : quality enhancement, gene pharming, boosting resistance to disease, and cost reduction.

72. Future of therapeutic cloning Advances in stem cell therapy. The availability of eggs. Learning to correct gene defects in inherited diseases.

73. Benefits of human cloning

74. Benefits of human cloning-1 Aging: possible to reverse the aging process. Cure for heart problems:. possible to treat heart attack victims by cloning their healthy heart cells and injecting them into the areas of the heart that have been damaged. Organ and tissue repair: Embryonic stem cells can be grown to produce organs or tissues to repair or replace damaged ones. Skin for burn victims, brain cells for the brain damaged, spinal cord cells for quadriplegics and paraplegics, hearts, lungs, livers, and kidneys could be produced. Cure for Incurable diseases: Conditions such as Alzheimer's disease, Parkinson's disease, diabetes, heart failure, degenerative joint disease, and other problems may be made curable.

75. Benefits of human cloning-2 5. Infertility: Human cloning could make it possible for infertile couples to have children . 6. Plastic, reconstructive, and cosmetic surgery: Instead of using materials foreign to the body for such procedures, doctors will be able to manufacture bone, fat, connective tissue, or cartilage that matches the patients tissues exactly.

76. Benefits of human cloning-3 7.Defective genes. possible to inactivate defective genes. 8.Down's syndrome. women at high risk for Down's syndrome can avoid that risk by cloning. 9.Tay-Sachs disease. This is an autosomal recessive genetic disorder could be prevented by using cloning.

77. Benefits of human cloning-4 10.liver failure. possible to clone livers for liver transplants. 11.kidney failure. possible to clone kidneys for kidney transplants. 12.leukemia . possible to clone the bone marrow for children and adults suffering from leukemia.

78. Benefits of human cloning-5 13.cancer. possible to switch cells on and off through cloning and thus be able to cure cancer. 14.cystic fibrosis. possible to produce effective genetic therapy against cystic fibrosis.

79. Benefits of human cloning-6 15.spinal cord injury. possible to grow nerves or the spinal cord back again when they are injured. 16.testing for genetic disease. Cloning technology can be used to test for and perhaps cure genetic diseases.

81. Thank you