Biotechnology of Mammalian Cloning Embryo Splitting Nuclear transplantation main technique in current cloning experiments Parthenogenesis only possible.
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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
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.
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.
• 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 (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
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