Animal Cloning

Animal Cloning

ES cells

Embryonic stem (ES) cells

Pluripotent stem cells derived from the inner cell mass of the blastocyst

Can be cultured manipulated and then reinjected into blastocysts where they can go on to contribute to all parts of embryo

In principle ES cells also might be ableto generatelarge quantities of any desired cell for transplantationinto patients

Human embryonic stem cells (hES cells)

bull hES cells represent the earliest stage of cells in human development

bull hES cells may differentiate into any one of the 230 different cells in human body

bull hES cells may treat many human diseases

Fertilized egg

Blastocyst ( 囊胚 )

hES cells

Ectoderm Mesoderm Endoderm

Skin Neural cells etcBone Muscle Blood cells etc

Digestive organs Lung etc

hES cell development stages

治疗多种疾病Treat many diseases


人的胚胎干细胞Human Embryonic Cells

Dopamine 神经原Dopamine neuronsDopamine 神经原Dopamine neurons 震颤性麻痹

Parkingsonrsquos disease

震颤性麻痹Parkingsonrsquos disease

胚状体细胞Embryoid Bodies

树状神经原Oligodendrocytes树状神经原Oligodendrocytes 脊髓损伤

Spinal cord injury

脊髓损伤Spinal cord injury

心肌细胞Cardiomyocytes心肌细胞Cardiomyocytes 心脏病

Heart disease

心脏病Heart disease

胰岛细胞Islet beta cells胰岛细胞Islet beta cells 糖尿病

Diabetes

糖尿病Diabetes

肝脏细胞Hepatocytes肝脏细胞Hepatocytes 肝脏疾病

Liver diseases

肝脏疾病Liver diseases

成骨细胞 Osteoblasts成骨细胞 Osteoblasts 骨质疏松症

Osteoporosis

骨质疏松症Osteoporosis

软骨细胞Chondrocytes软骨细胞Chondrocytes 骨关节炎

Osteoarthritis

骨关节炎Osteoarthritis

其他细胞Other cells其他细胞Other cells 其他疾病

Other Diseases

其他疾病Other Diseases

hES may differentiate into any human cells

Mouse embryonic stem cell cultures

LIF (leukaemia inhibitory factor) maintains embryonic stem cells

in an undifferentiated state

- LIF

ES cells spontaneously differentiate when allowed to aggregate

in the absence of LIF

Human stem cell lines available

httpwwwthe-funneled-webcomimagesEmbryonic20stem20cellsgif

(August 28 2001)

wwwlaskerfoundationorg newsweisestemcellhtml

Totipotent and pluripotent cells

Totipotent =meaning that its potential is total

pluripotent = they can give rise to many types of cells but not all types of cells (no fetus developed)

isolated directly from the inner cell mass of embryos at the blastocyst stage

(IVF-IT surplus embryos in case of humans)

Adult stem cells multipotent but not totipotent

More about stem cells

Embryonic stem cells Adult stem cells

Truly pluripotential More restricted pattern of differentiation

medical gain without ethical pain

several countries have sanctioned deriving

human ES-cell lines from lsquosurplusrsquo embryos

created through in vitro fertilization

Nuclear Transplantation

1 Enucleation of the cell

removal of the nucleus

From the a mature unfertilized oocyte (egg)

Or from the cell in quiescent state (inactive G0 phase of cell cycle) OR metaphase II

chromosomes are gently sucked out with a sharp micropipette

2 Nuclear transfer

A electrofusion

Nucleus comes from someone to be cloned

whole donor cell

injected beneath the zona pellucida

(the outer membrane of the oocyte)

and fusion of cells

induced by electrical impulses

B nuclear injection

naked nucleus

microinjected into cytoplastoocyte cytoplastENUCLEA

TION

Electrofusionhttpwwwbrinkmanncompdfcell_fusionpdf

Fusion induced by electric pulse

Cells brought close together

fusion pulse

Heterokaryon phase nuclei distinct

fusion product

Genetic Reprogramming

ldquode-differentiationrdquo ndash 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 ndash required for the nuclei from adult cells to develop normally

best completed in unfertilized oocytes (as plasma donors)

If cell for cloning taken from adult organism

Re-programming never achieved with same success as fertilization

Fig 5 from Nature Reviews Genetics 3 671

Development of the embryos from cell with ldquoalienrdquo nucleus

May be induced by chemical treatments

Developing embryos are grown in a culture to assess their viability

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

Therapeutic Cloning

1048663 1 Therapeutic cloning can only be justified by the utilitariancalculus that values potential medical treatments over thelives of the embryos who would be destroyed in order for theresearch to proceed However it is not ethical to sacrifice onehuman life for the real or potential benefit of others

1048663 2 It is unethical to view a human being - regardless of its age-as a means to an end Creation of human embryosspecifically for destructive research is opposed by ourcommunity and this is what is involved in therapeutic cloning

Why human Therateutic cloning is unethical in USA

1048663 3 Therapeutic cloning will undoubtedly lead to exploitation of womenIn order to create human clones for stem cell therapy an enormousnumber of womens eggs will need to be donated To do so womenmay be treated with superovulatory drugs and must undergo aninvasive procedure Complications may occur Advanced celltechnology paid up to $4000 to each woman who donated eggs fortheir cloning experiments It is likely that women of lower economicstatus will be exploited in this way

1048663 4 In addition to the ethical concerns above therapeutic cloning shouldbe banned because it increases the likelihood of reproductive cloningPreventing the implanting and subsequent birth of cloned embryosonce they are available in the laboratory will be impossible Already DrSeverino Antinori of Italy has plans to produce the first cloned humanThe most effective way to prevent reproductive cloning is to stop theprocess at the beginning with the creation of cloned embryos Sincethe overwhelming community consensus is that reproductive cloningshould be banned steps must be taken to ban therapeutic cloning aswell

10486635 Embryonic stem cell research and therapeutic cloning should also bebanned in view of the existence of other promising and ethicaltreatment options such as adult stem cell therapy (which has alreadybeen successfully used in patients)

10486636 In conclusion if destruction of excess embryos is allowed does thisjust apply to the embryos currently in storage or to future excessembryos as well The blatant irresponsibility of IVF clinics that haveallowed the accumulation of approximately 70000 embryos to date willhave no incentive to change if future excess embryos are also fodderfor the laboratory

1984 ndash A live lamb was cloned from sheep embryo cells

1986 ndash Early embryo cells were used to clone a cow

1993 ndash Calves were produced by transfer of nuclei from cultured embryonic cells1995 ndash Two sheep named Megan amp Morag were cloned using embryo cells

1996 ndash Birth of Dolly the first organism to be cloned from a fully differentiated adult cell1997 ndash Transgenic sheep named Polly was cloned containing a human gene

Mammal Cloning Timeline

httpwwwcnncom2001WORLDeurope0806clonecriticsindexhtml

Megan and Morag

Dolly

1998 ndash 50 mice were cloned in three generations from a single mouse

1998 ndash 8 calves were cloned from a single adult cow but only 4 survived to their first birthday1999 ndash A female rhesus monkey named Tetra was cloned by splitting early embryo cells2000 ndash Pigs and goats reported cloned from adult cells

2002 ndash Rabbits and a kitten reported cloned from adult cells

Tetra

httphshoustonisdorghspvaacademicScienceThinkquestgailtextbenefitshtml

Dolly

Dolly with her first newborn Bonnie

bull Born in July 1996 at the Roslin Institute in Scotland

bull First mammal to be cloned from an adult mammal using the nuclear transfer technique

bull 277 attempts were made before the experiment was successful

bullDolly 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 surrogate mother

Mammal Cloning allows propagation of endangered species

httpwwwhowstuffworkscomcloninghtmprintable

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

Comparison of Cloning Success Rates in Various

Animals Species

Number of oocytes used

Number of live

offspring

Notes

Mouse 2468 31 (13) -

Bovine 440 6 (14) 2 died

Sheep 417 14 (34) 11 died within 6 months

Pig 977 5 (05) -

Goat 285 3 (11) -

The table shows success rates of cloning when mature mammal cells were used

Yanagimachi R 2002 Cloning experience from the mouse and other animals Molecular and Cellular Endocrinology 21 March 187

Development and survival of cloned mouse embryos

Majority of the embryos die before and after implantation This figure shows that the present cloning technique is highly inefficient


Clone Birth Defects bull Cloned offspring often suffer from large offspring syndrome where the clone and the placenta that nourished it are unusually large

bull Cloned offspring often have serious inexplicable respiratory or circulatory problems which causes them to die soon after birth

bull Clones tend to have weakened immune systems and sometimes suffer from total immune system failure

bull Very few clones actually survive to adulthood 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

The whole story about cloning is not a reproductive story

The possibility of using cloning technology

to grow organs genetically identical

to our own for transplantation ndash

thereby avoiding rejection of foreign issues

httpmedlibmedutaheduWebPathCVHTMLCV001html

httpeasywebeasynetcouk~sflrlb3ajpg

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Embryonic stem (ES) cells

Pluripotent stem cells derived from the inner cell mass of the blastocyst

Can be cultured manipulated and then reinjected into blastocysts where they can go on to contribute to all parts of embryo

In principle ES cells also might be ableto generatelarge quantities of any desired cell for transplantationinto patients





Fertilized egg


hES cells













Spinal cord injury



Heart disease



Diabetes

糖尿病Diabetes


Liver diseases



Osteoporosis



Osteoarthritis



Other Diseases






- LIF





(August 28 2001)





















2 Nuclear transfer

A electrofusion


whole donor cell



and fusion of cells


B nuclear injection

naked nucleus


TION




fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Fertilized egg


hES cells













Spinal cord injury



Heart disease



Diabetes

糖尿病Diabetes


Liver diseases



Osteoporosis



Osteoarthritis



Other Diseases






- LIF





(August 28 2001)





















2 Nuclear transfer

A electrofusion


whole donor cell



and fusion of cells


B nuclear injection

naked nucleus


TION




fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Spinal cord injury



Heart disease



Diabetes

糖尿病Diabetes


Liver diseases



Osteoporosis



Osteoarthritis



Other Diseases






- LIF





(August 28 2001)





















2 Nuclear transfer

A electrofusion


whole donor cell



and fusion of cells


B nuclear injection

naked nucleus


TION




fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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- LIF





(August 28 2001)





















2 Nuclear transfer

A electrofusion


whole donor cell



and fusion of cells


B nuclear injection

naked nucleus


TION




fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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(August 28 2001)





















2 Nuclear transfer

A electrofusion


whole donor cell



and fusion of cells


B nuclear injection

naked nucleus


TION




fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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2 Nuclear transfer

A electrofusion


whole donor cell



and fusion of cells


B nuclear injection

naked nucleus


TION




fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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fusion pulse


fusion product














Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Therapeutic Cloning














Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Megan and Morag

Dolly




Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Tetra


Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Dolly











Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Animals Species


Number of live

offspring

Notes

Mouse 2468 31 (13) -



Pig 977 5 (05) -

Goat 285 3 (11) -


















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Animal Cloning

Documents

Transcript of Animal Cloning