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

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

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

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. GENETIC PRESERVATION Multiplication Of Elite Animals. Example…high milk producing dairy cow. Picture from Wells et al.

8. LOSSES & ABNORMALITIES EMBRYO CULTURE EFFECTS OOCYTE EFFECTS DONOR CELL EFFECTS DONOR CELL CULTURE EFFECTS HYDROALLANTOIS EXTENDED GESTATION KIDNEY BRAIN CARDIOVASCULAR MUSCLE SKELETAL INFERTILITY PLACENTAL PROBLEMS WITH SCNT 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. Oocyte Source Quality Maturation Process TECHNICAL METHODS Enucleation Nucleus transfer Activation Culture Conditions Cell Type Cell Isolate Culture Conditions Cell Cycle Stage Quality/selection of transferrable embryos RECIPIENT MANAGEMENT IMPRINT ERASURE OPTIMISATION AIDING ‘REPROGRAMMING’ MAXIMUM QUALITY OPTIMISING APPROACHES TO IMPROVE THE EFFICIENCY OF SCNT

13. SCNT EFFICIENCIES 2007 Cell LineTestNo Fused (% total) No Cleaved (% fused) No Blastocysts (% fused) No TransferredNo Live Births (% Trans) OP5DF3280224 (80.0)49 (17.5) 357 (20.0) SFF5+95 (100) 39 (41.0) 151 (6.7) LFF4+9490 (95.6) 32 (34.0) 151 (6.7) TOTAL+469 (92.1) 409 (87.2) 120 (25.6) 659 (13.8) OP5DF3-253 (82.4) 218 (71.1) 23 (9.0) 233 (13.0) SFF5-92 (94.8) 74 (80.4) 24 (26.1) 151 (6.7) LFF4-87 (85.3) 73 (83.9) 12 (13.8) 120 (0.0) TOTAL-432 (85.3) 365 984.5) 59 (13.7) 504 (8.0)

15. 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. Management of recipients has improved development, reduced the numbers of animals required and reduced the incidence of discomfort. Improvemnets in in vitro maturation and embryo culture have reduced the number of animals required and improved development.

16. 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