Hot Topics. Task: Answer the Questions. Who Are We? What Do We Do?

Transcription

1 Hot Topics Task: Answer the Questions Who Are We? What Do We Do?

2 We Are: The Reproductive Sciences Laboratory Department of Veterinary Physiology & Pharmacology, Texas A&M University Texas A&M AgriLife Research Interdisciplinary Faculty of Reproductive Biology Interdisciplinary Faculty of Genetics Department of Animal Sciences

3 Duane Kraemer Mark Westhusin Charles Long Michael Golding Approx. 100 M.S. and Ph.D. Students Numerous Veterinary and Undergraduate Students Approx. 300 Adult Education Students

4 What Do We Do? Animal Embryo Transfer: Cloning and Genetic Engineering

5 Embryo Transfer Movement of preimplantation embryos from the reproductive tract of the genetic mother (donor) to the reproductive tract of the surrogate mother (recipient).

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8 Objectives To increase the number of offspring from genetically valuable females. The end point of most other reproductive technologies such as: In-Vitro Fertilization Cloning

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11 First Records of Successful Egg Transfer Date Species 1891 rabbit 1932 goat 1933 rat 1933 sheep 1942 mouse 1949 goat 1951 cow 1951 pig 1963 quokka 1964 golden hamster Date Species 1968 ferret 1970 tammar wallaby 1974 horse 1975 mink 1976 baboon 1977 rhesus monkey 1978 man 1978 cat 1979 dog 1981 mongoban gerbil

12 First Embryo Transfer in a Livestock Species Warwick, B.L., R.O. Berry and W.R. Horlacher, Texas Agricultural Experiment Station Goats and Sheep

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16 First Purebred Embryo Transfer Calves Produced by a Commercial Company Born May 18, 1972 Two Simmental Heifers Livestock Breeders International, Inc.

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18 Granada Genetics

19 The Procedure

20 Superovulation

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22 Insemination 2-3 times during heat period

23 Collection of Embryos

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25 Location, Grading and Storage of Embryos

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35 Cryopreservation Direct Transfer Ethylene Glycol Propylene Glycol Voelkel & Hu, 1992

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37 Synchronization of Recipients

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39 Deposition of Embryos into Recipient Reproductive Tract

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44 Sheep and Goats

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47 In-Vitro Fertilization

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49 Horses

50 The First E T Horse in the United States

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53 Deer

54 Deer Embryos

55 First Deer ET Offspr Magyar, et al TAMU

56 Antelope

57 Suni Antelope E T Offspring

58 First Primate E T Offspring 1976

59 First Feline Embryo Transfer 1978

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63 Dogs

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67 First E T Dog 1979

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70 Reproductive Sciences Laboratory Dept. VTPP, Reproductive Sciences Complex, Highway 47 Approximately 3200 sq feet lab space (1/2 lab), 3200 sq feet office space, ots of equipment. Animal facilities Principal Investigators Mark Westhusin,Duane Kraemer, Charles Long, Michael Golding Grad Students, Postdocs and Staff Focus Basic vs applied research, many different animal models Evolution Embryo transfer -> in vitro embryo production -> animal cloning -> animal cloning and genetic engineering. History ET first cat, dog, non-human primate, white-tailed deer, other exotics, application in many other species (DCK). Cloning cattle, goats, first cat, first white tailed deer.

71 Current Projects at RSL Cloning Most efforts focused on using for producing transgenics Basic research to decipher developmental failure. Development and applications of genetic engineering and RNA interference Characterization of epigenetic control of gene expression during early development Development of new methods for contraception in animals.

72 Nuclear Transplantation With Embryonic Cells

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74 Micromanipulation

75 Micromanipulation

76 Enucleation

77 Recombination

78 Electrofusion

79 2-cell Cloned Dog Embryo

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81 Along Came Dolly Viable offspring derived from fetal and adult mammalian cells I. Wilmut, A. E. Schnieke *, J. McWhir, A. J. Kind * & K. H. S. Campbell Nature Feb 27;385(6619):810-3

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83 Ten years of clones. (Top to bottom) Wolf, muflon, African wild cat, dog, sheep, mule, domestic cat, buffalo, mouse, goat, rabbit, horse, gaur, cow, pig, rat, ferret. Jose Cibelli A Decade of Cloning Mystique Science 18 May 2007: Vol no. 5827, pp

84 Chance

85 Second Chance

86 Second Chance 2 Years Old

87 Bull 86

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90 Megan

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101 GM Farm Animals When Can I Order Green Eggs and Ham

102 Transgenic Animals Increased productivity growth, feed efficiency, milk, etc Disease resistance Novel products pharmaceuticals, nutriceuticals,etc Xenotransplantation

103 Methods for Genetic Engineering Pronuclear injection Virus mediated transfer Embryonic stem cells chimeras or cloning Electroporation Liposome injection Sperm mediated transfer

104 Embryo removed from adult animal

105 In the laboratory, new genetic material is introduced into the embryo.

106 Embryo with new genetic material transferred into adult animal

107 Transgenic offspring express desired gene Herds of genetically modified animals are created using natural breeding

108 Transgenic Mice

109 Transgenic pigs

110 Transgenic Pork Chops

111 Production of Pharmaceuticals in Transgenic Sheep

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113 Mammary Gland Advantages Major function is to secrete protein Easily obtained, inexpensive and renewable Not harmful to the animal Post translational modifications: glycosylation, gamma carboxylation High concentration of cells to 1,000 fold higher than tissue culture Inducible system Major proteins only found in milk 40mg/ml

114 Financial Advantage of Genetically Modified Animals $600 million to build/operate $3 million to build/operate This protein can be produced at either of these facilities in the same amounts. It represents a $200 million/year product in the pharmaceutical industry

115 The Beginning

116 Where are we now? Engineering cells and Cloning Transposons Zinc fingers Talens Lentiviral delivery

117 FugW vector with shrna targeting Prp

118 Proof of Concept

119 Targetting myostatin by RNAi Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. McPherron AC, Lawler AM, Lee SJ.

120 Double muscling in cattle due to mutations in the myostatin gene. McPherron AC, Lee SJ.

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122 Targeting Myostatin in Cattle

123 Last Few Years Long standing relationship with Texas A&M Collaboration was developed to continue to move the Tg Malaria program forward by reestablishing the founder line/herd of goats at Texas A&M Aim was to re-garner interest and ultimately support/funding to continue to move the program forward

124 Malaria is a Major World Health Problem Caused by protozoan parasites infection e.g. Plasmodium - Plasmodium falciparum ~80% all cases/ ~90% deaths - Plasmodium vivax - Plasmodium ovale - Plasmodium malariae Resides within liver and red blood cells hidden from immune system However, circulating, infected red blood cells are broken down by spleen High rates of morbidity and mortality, especially among children and women in endemic areas Incidence: million / year Death: million / year Control of malaria becoming increasingly difficult: Vector control: insecticide-resistance mosquito Chemotherapeutic agents: drug-resistant parasite Vaccine: candidate search (anti-sporozoite, anti-merozoite, transmission blocking). 124

125 Stowers, Chen, et al. PNAS Paper: Jan 2002 Confidential 125

126 GM goats encoding malaria antigen

127 Retroviral mediated transgenic animals 62 transgenic animals representing cattle (8), goats (12), sheep (24) and pigs (18). 1 year of work with vast majority of the time involving gestation. Risk assessment studies showed no movement of transgene to surrogate and no unwanted viral recombination (RCL)

128 Current Status of GM Livestock The ability to efficiently produce genetically modified livestock for food and/or the production of human medicines and other useful products is here. The applications of this technology and potential benefit to animal agriculture, animal and human health are mindboggling and limited only by our imagination.

129 How About a Kiss?