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

Nuclear TransplantationEnucleation 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.

Nuclear TransplantationEnucleation 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

Electrofusion http://www.brinkmann.com/pdf/cell_fusion.pdf

Fusion induced by electric pulse

Cells brought close together

fusion pulse

fusion product

Nuclear TransplantationEnucleation 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

Nuclear TransplantationEnucleation 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

Nuclear TransplantationEnucleation 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

Nuclear TransplantationEnucleation 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

Summary

oocyte cytoplastENUCLEATION

NUCLEARTRANSFERcytoplast clone cell

developing embryo in culture

INDUCTION OF EMBRYO

DEVELOPMENTgenetic reprogramming

embryo

IMPLANTATION

uterus of surrogate mother

Mammal Cloning

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

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 cells1995 – 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 cell1997 – Transgenic sheep named Polly was cloned containing a human gene

Mammal Cloning Timeline

http://www.cnn.com/2001/WORLD/europe/08/06/clone.critics/index.html

Megan and Morag

Dolly

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 birthday1999 – 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

Tetra

http://hs.houstonisd.org/hspva/academic/Science/Thinkquest/gail/text/benefits.html

Dolly

Dolly with her first newborn, Bonnie

• 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.

from Time Magazine, March 10, 1997

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.

Cloning Humans

http://www.cnn.com/2001/WORLD/europe/08/06/clone.doctor/index.html

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

http://science.howstuffworks.com/human-cloning.htm/printable

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.”

• 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

• Clonaid was founded by members of a religious sect called the Raelians

http://www.clonaid.com/

• 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

• 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

Problems with Cloning

 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.

Clones are Not Exact Copies

• Rainbow and Cc (cloned cat)

• Cloned cat hardly resembles her originalhttp://www.crystalinks.com/cloningcats.html

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

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.

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

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Telomeres.html

Pros and Cons of Cloning

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 formhttp://www.stanford.edu/dept/GYNOB/

rei/pics/scan9.tif

Pros

• learn about aging processese.g. the accelerated aging caused by shortened telomeres in clones like Dolly

http://www.swmed.edu/home_pages/cellbio/shay-wright/intro/gallery/sw_gallery.html

Pros

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

http://medlib.med.utah.edu/WebPath/CVHTML/CV001.html

Pros

• the preservation of endangered speciese.g. the Siberian Tiger

• Repair of genetic defects: in humans, inbred populations

http://www.photohome.com/photos/animal-pictures/wildlife/tiger-1.html

Pros

• Helping infertile couplesDr. Barbie, Ph.D. to the rescuePermitting the cloning of an individual so that they may enjoy the joys of parenting.

• Plastic, reconstructive and cosmetic surgery through the cloning of bodily tissuesDr. Barbie to the rescue againthe use of natural materials rather than objectionable and unsafe materials such as silicone

http://www.parenting.com/parenting/partners/happy_family/

Pros

• Engineered foods: genetically enhanced mammals can be cloned, bypassing the need for breedinge.g. cows that produce “humanized” milk

http://www.crazyforcows.com/cgi-mine/pscripts/gallery.cgi?which=crazyforCOWS

Cons (Drawbacks) of Cloning

• Clones being treated as less than human

• The creation of a Clone ArmyClones could me used as “cannon fodder” replacing traditional military recruitment tactics, disregarding their basic human rights

• Clones being harvested for their body parts

http://www.starwars.com/databank/organization/clonetroopers/index_eu.html

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?

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?

http://members.fortunecity.de/neutralzone/BorgCube03.htm

Cons• What if someone

attempted to clone a notorious figure such as: Adolf Hitler, Saddam Hussein, Osama bin Laden, Pol Pot, Genghis Khan

http://www.schoolshistory.org.uk/nazigermanyimages.htm

•Would cloning Einstein, Mozart, Newton, Gandhi or Jesus be more acceptable? http://www.cbc.ca/news/indepth/background/

cloning.html

The

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