1. CLONING MAMMALS

2. Biotechnology of Mammalian Cloning
Embryo Splitting
Nuclear transplantation
main technique in current cloning experiments
Parthenogenesis
only possible in females to give female progeny
still investigating – so far mostly failed attempts
earliest method of cloning
success limited to embryos split before implantation
Embryo splitting (40s and 50s) aka artificial twinning
First done in 1885 with sea urchines
Mammals – first done in 1995 – seperate cells in developing embryos, let each cell multiply and develop into an embryo on its own, each embryo genetically identical
Parthenogenesis (testing now – not been successful with mammals)
Diploid precursor cells (which undergo meiosis to become oocytes) were mechanically, electrically, or chemically induced to develop as if it was a fertilized oocyte. Abnormal differentiation - No known embryos survive, but good for stem cells derivation – imprinting/division of labour between maternal/paternal genome???
Nuclear transplantation – aka somatic cell nuclear transfer (SCNT)
First done with frogs in 1952
Then 1975 rabbits – first mammal with NT (with embryonic somatic cell) then sheep (1986) and cow (1987)
In 1996 – first mammal with NT from ADULT somatic cell (Dolly!!)

3. Nuclear Transplantation
Enucleation of donor cell
removal of the nucleus
enucleated cell = cytoplast
donor preferably:
a mature unfertilized oocyte (egg)
in inactive state (inactive G0 phase of cell cycle)
chromosomes are gently sucked out with a sharp micropipette, removing the nucleus as a whole.
Donor cell – preferably a in vivo-matured unfertilized oocyte, but can also use a newly fertilized zygote
quiescent state – can be induced by starvation – depriving cells of nutrients – cells stop dividing – thought to help genetic reprogramming of the new embryo
Metaphase II – when all the chromosomes are aligned along the equator – easier to suck out all the chromosomes at once without leaving much behind
When DNA sucked out – plasma membrane reseal itself around the tip, leaving cytoplast intact

4. Nuclear Transplantation
Enucleation of donor cell
Nuclear Transfer
the nucleus of the individual to be cloned is transferred to the cytoplast in one of the 2 ways:
1) electrofusion – whole nucleus donor cell injected beneath the zona pellucida (the outer membrane of the oocyte) and fusion of cells induced by electrical impulses
2) nuclear injection – naked nucleus microinjected into cytoplast
Electric shock to mimic effect of sperm fusion during fertilization
Nuclear injection more successful

5. Electrofusion Fusion induced by electric pulse
fusion pulse
Cells brought close together
fusion product
Fusion induced by electric pulse

6. Nuclear Transplantation
Enucleation of donor cell
Nuclear Transfer
the nucleus donor could be obtained from a developing embryo, or a fully-grown adult
Is there a difference?
cells from embryo are undifferentiated
early embryonic cells are totipotent – they have the potential to develop into all types of differentiated cells
adult cells are differentiated
Genomic DNA rearranged during differentiation – only some types of differentiated cells can be used as nuclear donor
RBC can’t – no nuclei!

7. Nuclear Transplantation
Enucleation of donor cell
Nuclear Transfer
Genetic Reprogramming
“de-differentiation” – rearranging the genome of the nucleus to restore its totipotency so it can differentiate into different types of cells and develop into a whole organism
must occur after nuclear transfer to successfully produce the clone – required for the nuclei from adult cells to develop normally
best completed in unfertilized oocytes
Mechanism of genetic reprogramming still unclear – under intensive study
Genetic reprogramming – rearranging the DNA back to what it was before differentiation – required to express genes essential in embryonic development – thought to be aided by proteins present in eggs/zygote
Of course undifferentiated embryonic cells yield higher success rate!
Imcomplete genetic reprogramming lead to low cloning efficiency
Complication – imprinting!

8. Nuclear Transplantation
Enucleation of donor cell
Nuclear Transfer
Genetic Reprogramming
Embryonic Development
may be induced by chemical treatments
developing embryos are grown in a culture to assess their viability
Embryonic development can be chemically activated, usually with a combination of an ionophore and a protein kinase inhibitor or by microinjection of nitric oxide.

9. Nuclear Transplantation
Enucleation of donor cell
Nuclear Transfer
Genetic Reprogramming
Embryonic Development
Implantation of Embryo
embryos are surgically transferred into the uteri of suitable surrogate mothers
many embryos are transferred to each surrogate mothers to ensure implantation
Embryonic development can be chemically activated, usually with a combination of an ionophore and a protein kinase inhibitor or by microinjection of nitric oxide.

10. Summary ENUCLEATION oocyte cytoplast NUCLEAR TRANSFER cytoplast
clone cell

11. INDUCTION OF EMBRYO DEVELOPMENT genetic reprogramming
developing embryo in culture
IMPLANTATION
embryo
uterus of surrogate mother

12. Mammal Cloning
January 8, 2001 Noah, a baby bull gaur, became the first clone of an endangered animal.

13. Mammal Cloning Timeline
Megan and Morag
1984 – A live lamb was cloned from sheep embryo cells
1986 – Early embryo cells were used to clone a cow
1993 – Calves were produced by transfer of nuclei from cultured embryonic cells
1995 – Two sheep, named Megan & Morag, were cloned using embryo cells
1996 – Birth of Dolly, the first organism to be cloned from a fully differentiated adult cell
1997 – Transgenic sheep named Polly was cloned containing a human gene
Dolly

14. 1998 – 50 mice were cloned in three generations from a single mouse
Tetra
1998 – 50 mice were cloned in three generations from a single mouse
1998 – 8 calves were cloned from a single adult cow, but only 4 survived to their first birthday
1999 – A female rhesus monkey named Tetra was cloned by splitting early embryo cells.
2000 – Pigs and goats reported cloned from adult cells
2002 – Rabbits and a kitten reported cloned from adult cells

15. Dolly Born in July 1996 at the Roslin Institute in Scotland
First mammal to be cloned from an adult mammal using the nuclear transfer technique
277 attempts were made before the experiment was successful
Dolly died in February 14, 2003 of progressive lung disease at the age of 6; whereas normal sheep can live up to 12 years of age.
Dolly with her first newborn, Bonnie

16. from Time Magazine, March 10, 1997

17. Comparison of Cloning Success Rates in Various Animals
Species
Number of oocytes used
Number of live offspring
Notes
Mouse
2468
31 (1.3%) -
Bovine
440
6 (1.4%)
2 died
Sheep
417
14 (3.4%)
11 died within 6 months
Pig
977
5 (0.5%)
Goat
285
3 (1.1%)
The table shows success rates of cloning when mature mammal cells were used.

18. Cloning Humans

19. Scientists plan to use cell nuclear transfer, the same procedure that was used to create Dolly

20. Clonaid Human Cell Embryos http://www.clonaid.com/
“The main goal of CLONAID™ is to offer reproductive human cloning on a worldwide basis to infertile couples, homosexual couples, people infected with the HIV virus as well as to families who lost a beloved family member.”

21. Clonaid was founded by members of a religious sect called the Raelians
Clonaid was founded by members of a religious sect called the Raelians
On Thursday, December 26, 2002, Clonaid claimed to have successfully created the first human clone, a 7 pound baby girl named Eve
Eve was created using DNA from her mother’s skin cells and is a genetic twin of her mother, a 31-year-old American citizen

22. After originally stating that a panel of independent scientists would be allowed to verify that Eve was a clone of her mother, on January 3, 2003 it was announced that no tests would be performed to prove Eve’s existence or that she was a clone
Clonaid now claims that 5 cloned humans have been born, the most recent being born February 4, 2003
There is still no proof that a human clone has ever been born
Many scientists believe that the Clonaid claim is a hoax

23. Problems with Cloning Clones are not exact copies
· The cloning process is not safe
· Cloned offspring often suffer from birth defects
The clones that survive to adulthood seem to age faster than normal

24. Clones are Not Exact Copies
· A clone’s mitochondrial DNA comes from the donor egg cell. Unless the donor egg comes from the person being cloned their mitochondrial DNA will be different.
· Random developmental events, such as X-chromosome inactivation, can cause clones to differ greatly in appearance.
· Different environmental and social factors can cause clones to vary greatly in both appearance and personality.
· No clone will ever be exactly the same as the original.
Rainbow and Cc (cloned cat)
Cloned cat hardly resembles her original

25. Cloning is Not Safe
The success rate of an implanted clone embryo actually producing a cloned offspring is very low.
The actual mechanical process involved in cloning somehow causes developmental problems in clones.
Any clones that survive until birth often suffer from birth defects.
Dolly (left) and her surrogate mother
It took 277 attempts before Dolly became the first cloned sheep

26. Clone Birth Defects
Cloned offspring often suffer from large offspring syndrome, where the clone and the placenta that nourished it are unusually large.
Cloned offspring often have serious inexplicable respiratory or circulatory problems, which causes them to die soon after birth.
Clones tend to have weakened immune systems and sometimes suffer from total immune system failure.
Very few clones actually survive to adulthood.

27. Clone Aging Clones appear to age faster than normal.
Clones experience problems associated with old age, such as arthritis, while they are still young.
This may be due to the fact that clones have shorter telomeres (areas on the ends of chromosomes that shorten with age) than non-clones of the same age.
An example of a chromosome

28. Pros and Cons of Cloning

29. Pros (Benefits) of Cloning
learning about embryonic development and the differentiation of tissues
Better understanding of the factors needed for a properly functioning fetus and a normal individual to form

30. Pros learn about aging processes
e.g. the accelerated aging caused by shortened telomeres in clones like Dolly

31. Pros
The possibility of using cloning technology to grow organs genetically identical to our own for transplantation – thereby avoiding rejection issues

32. Pros the preservation of endangered species e.g. the Siberian Tiger
Repair of genetic defects: in humans, inbred populations

33. Pros Helping infertile couples Dr. Barbie, Ph.D. to the rescue
Permitting the cloning of an individual so that they may enjoy the joys of parenting.
Plastic, reconstructive and cosmetic surgery through the cloning of bodily tissues
Dr. Barbie to the rescue again
the use of natural materials rather than objectionable and unsafe materials such as silicone

34. Pros
Engineered foods: genetically enhanced mammals can be cloned, bypassing the need for breeding
e.g. cows that produce “humanized” milk

35. Cons (Drawbacks) of Cloning
Clones being treated as less than human
The creation of a Clone Army
Clones could me used as “cannon fodder” replacing traditional military recruitment tactics, disregarding their basic human rights
Clones being harvested for their body parts

36. Cons The pressing question: What if clones don’t have a soul???
Cloning is a trial-and-error process: many embryos fail to develop and this can be interpreted as murder by some.
Would it be ethical to bring a human clone into the world given the safety concerns, especially the health problems experienced by clones produced thus far?

37. Cons Would certain individuals seek immortality?
What would be said of our individuality, when any of us can be duplicated at will?
What if we all became mindless automatons?

38. Cons
What if someone attempted to clone a notorious figure such as: Adolf Hitler, Saddam Hussein, Osama bin Laden, Pol Pot, Genghis Khan
Would cloning Einstein, Mozart, Newton, Gandhi or Jesus be more acceptable?

39. The
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