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CLONING FARM ANIMALS

CLONING FARM ANIMALS. Keith H.S.Campbell. School of Biosciences University of Nottingham UK. 2. Donor Cells Fresh or Cultured. 3. Enucleation. 4. Transfer donor nucleus. 5. Activation. 6. Culture. 7. Transfer to surrogate. Gestation. THE PROCESS OF SOMATIC

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CLONING FARM ANIMALS

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  1. CLONING FARM ANIMALS Keith H.S.Campbell School of Biosciences University of Nottingham UK

  2. 2. Donor Cells Fresh or Cultured 3. Enucleation 4. Transfer donor nucleus 5. Activation 6. Culture 7. Transfer to surrogate Gestation THE PROCESS OF SOMATIC CELL NUCLEAR TRANSFER • Unfertilised egg/MII oocyte • Fresh or usually from abbatoir

  3. DOLLY 1996

  4. NUCLEAR TRANSFER FROM SOMATIC CELLS: • Megan & Morag • Born July 1995 • 1st mammals produced by NT from a cultured differentiated cell line • Quiescent cells used as nuclear donors

  5. SUCCESSES OF SCNT • GAUR 2000 • MULE 2003 • WOLF 2007 • SHEEP 1996 • MOUFLON 2001 • DEER 2003 • CATTLE 1998 • CAT 2002 • HORSE 2003 • MICE 1998 • CAMEL 2009 • RABBIT 2002 • DOG 2005 • BANTENG 2003 • GOATS 1999 • IBEX 2009 • RAT 2003 • FERRET 2006 • PIGS 2000

  6. ROLE OF CLONING IN AGRICULTURE • SELECTIVE BREEDING TO PRODUCE GOOD QUALITY ANIMALS • WITH DESIRED TRAITS • LIMITED BY NUMBER OF ANIMALS AVAILABLE • IN PARTICULAR THE NUMBER OF FEMALES • CLONING ALLOWS THE MULTIPLICATION OF ELITE ANIMALS • FOR DISSEMINATION OD GENETIC TRAITS, ALSO ALLOWS THE • PRODUCTION OF BREEDING ANIMALS FROM AGED OR DISEAESED • POPULATIONS • CLONING ALLOWS PRESERVARTION OF PHENOTYPES

  7. GENETICPRESERVATION Multiplication Of Elite Animals. • Example…high milk producing dairy cow. • Picture from Wells et al.

  8. PROBLEMS WITH SCNT LOSSES & ABNORMALITIES • EMBRYO CULTURE EFFECTS • OOCYTE EFFECTS • DONOR CELL EFFECTS • DONOR CELL CULTURE EFFECTS • HYDROALLANTOIS • EXTENDED GESTATION • KIDNEY • BRAIN • CARDIOVASCULAR • MUSCLE • SKELETAL • INFERTILITY • PLACENTAL INCOMPLETE OR INEFFICIENT REPROGRAMMING

  9. CLONING DOLLY AND EARLY OVINE CLONING • Oocytes, in-vivo matured, superovulated ewes. • Oocyte age approx 36hpGnRH. • Enucleation – physical, sharp glass pipette. • Fusion, electrical. • Activation, same pulse as for fusion. • Culture in-vivo, ligated oviduct. OBJECTIONS: Use of animals for oocyte collection and culture.

  10. PRESENT SHEEP CLONING PROTOCOL • In vitro oocyte maturation. • Modified physical enucleation protocol. • Electrical Fusion. • Chemical activation (ionomycin + CHX). • In-vitro culture (SOFM).

  11. RECLONING DOLLY 2006 • In-vitro. • 11 Blastocysts. • 8 Recipients. • 1 Live Lamb. • 1/11 = 9%. • Original = 3.45% CAN WE IMPROVE EFFICIENCY ?

  12. APPROACHES TO IMPROVE THE EFFICIENCY OF SCNT TECHNICAL METHODS Enucleation Nucleus transfer Activation Culture Conditions Oocyte Source Quality Maturation Process Quality/selection of transferrable embryos OPTIMISATION AIDING ‘REPROGRAMMING’ MAXIMUM QUALITY RECIPIENT MANAGEMENT Cell Type Cell Isolate Culture Conditions Cell Cycle Stage OPTIMISING IMPRINT ERASURE

  13. SCNT EFFICIENCIES 2007

  14. SUMMARY • SCNT is a process involving multiple procedures. Each of these • procedures may have effects on subsequent embryo and foetal • development. • Significant research has reduced the incidence of abnormalities • and improved the frequency of successful development. • Improvemnets in in vitro maturation and embryo culture have • reduced the number of animals required and improved development. • Management of recipients has improved development, reduced • the numbers of animals required and reduced the incidence of • discomfort.

  15. COMMON QUESTIONS • Did Dolly age/die prematurely ? • No…..retroviral lung disease • Do other clones age prematurely • No…normal lifespan • Is there any danger from consumption of cloned • animals or their progeny ? • Significant research has shown no danger. • FDA / FSA/ EFSA

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