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Abnormalities In Cloned Animals

Abnormalities In Cloned Animals . Megan Shoemaker Anna Leppien October 25, 2006. History . 1938: concept of a nuclear transfer 1952: first success with amphibians 1982: first mouse clone 1997: Dolly. Cloning by Nuclear Transfer . Edwards et al. 2003. Developmental Abnormalties

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Abnormalities In Cloned Animals

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  1. Abnormalities In Cloned Animals Megan Shoemaker Anna Leppien October 25, 2006

  2. History • 1938: concept of a nuclear transfer • 1952: first success with amphibians • 1982: first mouse clone • 1997: Dolly

  3. Cloning by Nuclear Transfer Edwards et al. 2003

  4. Developmental Abnormalties • Mortality • Prenatal Development • Postnatal Development • Causes • Placental Malformation • Somatic Cell Reprogramming • Telomeres

  5. Statistics • High levels of Bias • Incidence Lower than Publicized • High Variations in Reports • Methods • Cell source • Species • Goal of Report

  6. Mortality • Live Births • Kato et al. : 14% (calves) • Amano et al.: 1-3% (mice) • Solter : 3% (overall) • Chavatte-Palmer et al. : 2-10% (calves) • Rideout et al.: 1-10% (overall)

  7. Mortality… %“Normal” Offspring Cibelli et al. 2002

  8. Mortality • Success is affected by source cell type • Oviduct, cumulus cell: highly successful • Skin and ear: 2 of 10 embryos survive • Programming of Donor Nuclei • Species Differences Tsunoda, Y and Y. Kato. 2002.

  9. Mortality… Periods of Clone Loss • 1. Preimplantation • > 65% of blastocysts fail • 2. Day 30 to Day 60 • Death of >50% of embryos • Vs. Natural Breeding: 2-10% IVF: 16% Edwards et al. 2003

  10. Mortality… Periods of Clone Loss • 3. Second Trimester • Spontaneous abortion • Some abnormalities • Abnormal placenta • Low cotyledon count • Thickened fetal membranes Edwards et al. 2003

  11. Mortality… Periods of Clone Loss 4. Third Trimester • Hydrallantois • Reduction in placentomes • Adema of placental membranes • Hypertrophy of cotyledons • Adema of Umbillicus Edwards et al. 2003

  12. Mortality… Periods of Clone Loss • 5. Postnatal • Large birth size • Inadequate Organ Development • Lung dysmaturity • Repiratory distress • Metabolic acidosis Edwards et al. 2003

  13. Prenatal Development • General retarded development • Abnormal Growth Curve • Cattle: abnormal mRNA by blastocyst stage

  14. Prenatal Development… Overall Growth Altered growth curve Hill et al. 2000

  15. PrenatalDevelopment… Overall Growth Low crown-rump length Increased Body Weight Increased Organ Weights - kidney - heart - liver

  16. Prenatal Development… Overall Growth Hill et al. 2000

  17. Prenatal Development… Overall Growth Normal Mouse NT Mouse Eggan et al. 2001

  18. Prenatal Development… Placental Insufficiencies • Classic Pathology of NT fetuses • Abnormal • Poor Development • Low Vasculature • Cotelydons • High individual weights • Low total number • High overall weight

  19. Prenatal Development… Placental Insufficiencies • Deficiencies of Feto-Maternal Exchange • Slow initial Development • Compensation later in gestation

  20. Prenatal Development… Placental Insufficiencies De Sousa et al. 2001

  21. Prenatal Development… Cardiac Effects • Increased Heart Rate • Normal until day 37 (cattle) • Dramatic Increase after day 44 Bertolini et al. 2002

  22. Prenatal Development… Cardiac Effects • Increased Blood Pressure • Increased Organ Weight • Day 100 (cattle) • AI: .16 g • IVP: .30 g • NT: 1.08g Lee et al. 2004

  23. Prenatal Development… Liver Effects • Bloodless • Pale • Increased Organ Weight • Day 150 (cattle) • AI: 113 g • IVP: 105 g. • NT: 159 g. De Sousa et al. 2001 Lee et al. 2004

  24. Prenatal Development… Cerebral Effects • Accumulation of Fluid in Fourth Ventricle • Dermal Hemorrhage • Behavioral Problems • Mental Retardation? Lee et al. 2004

  25. Prenatal Development… Cerebral Effects De Sousa et al. 2001

  26. Postnatal Development • Large Birth Size • Various Health Problems • Decreased Postnatal Survival Rate • Range of outcomes • Normal/healthy  Fatally Ill

  27. Postnatal Development… Large Offspring Syndrome • Cattle, Sheep, Mice • Associated with in vitro embryos • Exacerbated in NT fetuses • Lee et al. 2004 – cattle • 17% increase in birth weight • In vivo: 34.4 kg • NT: 40.4 kg • Growth curves normalize after birth Farin et al. 2006

  28. Postnatal Development… Large Offspring Syndrome NT: 55.1 kg IVP: 45.7 kg AI: 43.7 kg Chavatte-Palmer, et al. 2002

  29. Postnatal Development… Health Problems • Wide Variety of Problems • Many manifest well after birth • Cattle: • Immature Lungs • Hypoglycemia • Metabolic Acidosis • Pulmonary Hypertension

  30. Postnatal Development… Health Problems http://www.geocities.com/uconnyanglab/

  31. Postnatal Development… Health Problems • Mice • Kidneys • Hypertrophy • Increased weight • Lesions • Enlarged renal pelvis • Distension of renal veins • Necrosis of Glomerulus • Abnormalities being in young adults Shimozawa et al. 2006

  32. Postnatal Development… Reproduction • Varied Reports • Calves (Tian et al.) • Late puberty • Reproduction otherwise normal June 3, 2001 “Norm” http://www.geocities.com/uconnyanglab/

  33. Postnatal Development… Reproduction • Mice • Aged mice (>20 months) • No spermatogenesis • Atrophied testes • Normal Testis Function as young adults • Progeny Test • Normal offspring - imprinting reset in gametogenesis Shimozawa et al. 2006

  34. Postnatal Development… Reproduction

  35. Postnatal Development… Lifespan • Conclusions are Premature • Early Death? • Livestock: difficult to determine • Mice: early death reported • Pneumonia • Hepatic Failure

  36. Postnatal Development… Lifespan Ogonuki et al. 2002

  37. Causes of Abnormalities • Placental malformation • Somatic Cell Reprogramming/DNA methylation • Telomere length

  38. Placental Malformation • Avasular or hypovascular • Altered expression of CDKN1 and TP73 • Affect expression of VEGF • Reduced number of cotyledons • Most striking in fetuses arrested at day 28 or earlier • Cotyledons enlarged • compensatory Farin et al.

  39. Placental Malformation De Sousa et al

  40. Placental Malformation… Fetal Edema • Vascular deficiencies • Fluid osmotically drawn from maternal to fetal circulation • Enlarged umbilical vessels and membranes • Hydrallantois De Sousa et al

  41. Placental Malformation Placental Malformation… Fetal Edema • Enlargement of liver • Site of high metabolism • Metabolites build up • Dermal hemorrhaging • Swelling of brain structures • Fourth ventricle De Sousa et al

  42. Somatic Cell Reprogramming • Somatic cell must undifferentiate into pluripotent embryo cells • Then redifferentiate into somatic cells • UNNATURAL PROCCESS!!

  43. Somatic Cell Reprogramming/DNA Methylation • Dnmt1 protein –regulates DNA methylation pattern • Dnmt1o – oocyte derived – enters nucleus at eight cell stage • Dnmt1s – somatic cell derived – enters nucleus after implantation Chung et al

  44. Somatic Cell Reprogramming… Dnmt1 Protein • Disruption of Dnmt1o and Dnmt1s in cloned embryos • In eight-cell-stage • No Dnmt1o in nucleus • Normal amount in cytoplasm • Dnmt1s expressed in blastomeres De Sousa et al

  45. Somatic Cell Reprogramming… Dnmt1s expression in cloned and normal embryos A: Dnmt1s in cloned embryo B: Dnmt1s in normal, fertilized eggs Chung et al

  46. Somatic Cell Reprogramming… Dnmt1 • Absence of Dnmt1o in nucleus of 8-cell embryo • Inappropriate expression of Dnmt1s • Unable to maintain methylation patterns on alleles of imprinted genes Chang et al

  47. Telomeres • Structures at end of chromosomes • Contain proteins and DNA • Protect chromosomes

  48. Telomeres • Shorten as cells divide • Shortened telomeres may be a sign for cells to stop dividing • Biological clock for cellular aging Xu et al

  49. Telomeres… Dolly • Telomere length • One year: 19 kb vs. 24 kb in control sheep • Donor cells: 6 year sheep mammary gland epithelial cells Xu et al

  50. Telomeres Telomeres… Other studies • Telomere length same as or longer than age matched controls • Same in cloned cattle (Tian, et. al.) • Longer in cloned cattle (Lanza, et. al.) • Same in 6 generations of mice (Wakayama et. al.)

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