CLONING FARM ANIMALS

Keith H.S.Campbell

School of BiosciencesUniversity of Nottingham

UK

1. Unfertilised egg/MII oocyte Fresh or usually from abbatoir

3. Enucleation

4. Transfer donor nucleus

Gestation 7. Transfer to surrogate

THE PROCESS OF SOMATICCELL NUCLEAR TRANSFER

6. Culture

2. Donor CellsFresh or Cultured

5. Activation

DOLLY

1996

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

•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

ROLE OF CLONINGIN 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

GENETIC PRESERVATION Multiplication Of Elite Animals.

• Example…high milk producing dairy cow.

• Picture from Wells et al.

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 ORINEFFICIENT REPROGRAMMING

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.

PRESENT SHEEP CLONING PROTOCOL

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

RECLONING DOLLY 2006

• In-vitro.• 11 Blastocysts.• 8 Recipients.• 1 Live Lamb.• 1/11 = 9%.• Original = 3.45%

CAN WE IMPROVE EFFICIENCY ?

Oocyte Source Quality

Maturation Process

TECHNICAL METHODS

EnucleationNucleus transfer

ActivationCulture Conditions

Cell TypeCell IsolateCulture ConditionsCell Cycle Stage

Quality/selection of transferrable embryos

RECIPIENTMANAGEMENT

IMPRINT ERASURE

OPTIMISATIONAIDING ‘REPROGRAMMING’

MAXIMUM QUALITY

OPTIMISING

APPROACHES TO IMPROVE THE EFFICIENCY OF SCNT

SCNT EFFICIENCIES 2007Cell Line Test No Fused

(% total)

No Cleaved (% fused)

No Blastocysts(% fused)

No Transferred No Live Births

(% Trans)

OP5DF3 280 224 (80.0) 49(17.5)

35 7(20.0)

SFF5 + 95 95(100)

39(41.0)

15 1(6.7)

LFF4 + 94 90(95.6)

32(34.0)

15 1(6.7)

TOTAL + 469(92.1)

409(87.2)

120(25.6)

65 9(13.8)

OP5DF3 - 253(82.4)

218(71.1)

23(9.0)

23 3(13.0)

SFF5 - 92(94.8)

74(80.4)

24(26.1)

15 1(6.7)

LFF4 - 87(85.3)

73(83.9)

12(13.8)

12 0(0.0)

TOTAL - 432(85.3)

365984.5)

59(13.7)

50 4(8.0)

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.

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