Abnormalities In Cloned Animals History Cloning by Nuclear Transfer StatisticsMortality %“Normal” OffspringMortalityPeriods of Clone LossPeriods of Clone LossPeriods of Clone LossPeriods of Clone LossPrenatal DevelopmentOverall GrowthOverall GrowthOverall GrowthOverall GrowthPlacental InsufficienciesPlacental InsufficienciesPlacental InsufficienciesCardiac EffectsCardiac EffectsLiver EffectsCerebral EffectsCerebral EffectsPostnatal DevelopmentLarge Offspring SyndromeLarge Offspring SyndromeHealth ProblemsHealth ProblemsHealth ProblemsReproductionReproductionReproductionLifespanLifespanCauses of AbnormalitiesPlacental MalformationPlacental MalformationFetal EdemaFetal EdemaSomatic Cell ReprogrammingSomatic Cell Reprogramming/DNA MethylationDnmt1 ProteinDnmt1TelomeresTelomeresDollyOther studiesOther studiesThe Big Picture!! More Work Needs to be Done!Questions?Overall GrowthPlacental InsufficienciesmplicationsAbnormalities In Cloned AnimalsMegan ShoemakerAnna LeppienOctober 25, 2006History• 1938: concept of a nuclear transfer• 1952: first success with amphibians• 1982: first mouse clone• 1997: DollyCloning by Nuclear Transfer Edwards et al. 2003• Developmental Abnormalties– Mortality– Prenatal Development– Postnatal Development•Causes– Placental Malformation– Somatic Cell Reprogramming– TelomeresStatistics• High levels of Bias• Incidence Lower than Publicized• High Variations in Reports– Methods– Cell source– Species– Goal of ReportMortality• 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)%“Normal” OffspringCibelli et al. 2002Mortality…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 DifferencesTsunoda, Y and Y. Kato. 2002.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%Mortality…Edwards et al. 2003Periods of Clone Loss• 3. Second Trimester– Spontaneous abortion– Some abnormalities• Abnormal placenta• Low cotyledon count• Thickened fetal membranesMortality…Edwards et al. 2003Periods of Clone Loss4. Third Trimester– Hydrallantois• Reduction in placentomes• Adema of placental membranes• Hypertrophy of cotyledons– Adema of UmbillicusMortality…Edwards et al. 2003Periods of Clone Loss• 5. Postnatal– Large birth size– Inadequate Organ Development• Lung dysmaturity• Repiratory distress• Metabolic acidosisMortality…Edwards et al. 2003Prenatal Development• General retarded development• Abnormal Growth Curve• Cattle: abnormal mRNA by blastocyst stageOverall GrowthAltered growth curvePrenatal Development…Hill et al. 2000Overall GrowthLow crown-rump lengthIncreased Body WeightIncreased Organ Weights- kidney-heart- liverPrenatal Development…Overall GrowthPrenatal Development…Hill et al. 2000Overall GrowthPrenatal Development…Normal Mouse NT MouseEggan et al. 2001Placental Insufficiencies• Classic Pathology of NT fetuses• Abnormal• Poor Development• Low Vasculature• Cotelydons– High individual weights– Low total number– High overall weightPrenatal Development…Placental Insufficiencies• Deficiencies of Feto-Maternal Exchange• Slow initial Development• Compensation later in gestationPrenatal Development…Placental InsufficienciesPrenatal Development…De Sousa et al. 2001Cardiac Effects• Increased Heart Rate– Normal until day 37 (cattle)– Dramatic Increase after day 44Prenatal Development…Bertolini et al. 2002Cardiac Effects• Increased Blood Pressure• Increased Organ Weight– Day 100 (cattle)• AI: .16 g• IVP: .30 g• NT: 1.08gPrenatal Development…Lee et al. 2004Liver Effects• Bloodless •Pale• Increased Organ Weight– Day 150 (cattle)• AI: 113 g• IVP: 105 g.• NT: 159 g.Prenatal Development…De Sousa et al. 2001Lee et al. 2004Cerebral Effects• Accumulation of Fluid in Fourth Ventricle• Dermal Hemorrhage• Behavioral Problems• Mental Retardation?Prenatal Development…Lee et al. 2004Cerebral EffectsPrenatal Development…De Sousa et al. 2001Postnatal Development• Large Birth Size• Various Health Problems• Decreased Postnatal Survival Rate– Range of outcomes• Normal/healthy ÅÆ Fatally IllLarge 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 birthPostnatal Development…Farin et al. 2006Large Offspring SyndromePostnatal Development…Chavatte-Palmer, et al. 2002NT: 55.1 kgIVP: 45.7 kgAI: 43.7 kgHealth Problems• Wide Variety of Problems– Many manifest well after birth• Cattle:– Immature Lungs– Hypoglycemia– Metabolic Acidosis– Pulmonary HypertensionPostnatal Development…Health ProblemsPostnatal Development…http://www.geocities.com/uconnyanglab/Health Problems•Mice– Kidneys• Hypertrophy• Increased weight•Lesions– Enlarged renal pelvis– Distension of renal veins• Necrosis of Glomerulus• Abnormalities being in young adultsPostnatal Development…Shimozawa et al. 2006Reproduction• Varied Reports• Calves (Tian et al.)– Late puberty– Reproduction otherwise normalPostnatal Development…June 3, 2001“Norm”http://www.geocities.com/uconnyanglab/Reproduction•Mice – Aged mice (>20 months)• No spermatogenesis• Atrophied testes– Normal Testis Function as young adults– Progeny Test• Normal offspring- imprinting reset in gametogenesisPostnatal Development…Shimozawa et al. 2006ReproductionPostnatal Development…Lifespan• Conclusions are Premature• Early Death?– Livestock: difficult to determine– Mice: early death reported• Pneumonia • Hepatic Failure Postnatal Development…LifespanPostnatal Development…Ogonuki et al. 2002Causes of Abnormalities• Placental malformation• Somatic Cell Reprogramming/DNA methylation• Telomere lengthPlacental 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– compensatoryFarin et al.Placental