Keys to Science, August 2004Keys to Science, August 2004

•Ditto

•Gene

•Neti

•Cumulina

•Megan, & Morag

•Idaho Gem

Who areWho are

Dolly & Mom, 1997Dolly & Mom, 1997

??

Cloning is a novel form of sexual reproduction.

OR

Cloning is a novel form of asexual reproduction.

??

??Clones are produced only in

laboratories.

OR

Clones are produced as a normal part of an organism’s biology.

RV

G0

RVRV

&

Normal CloningNormal Cloning

?

Bottom lineBottom line:

Cloning Time LineCloning Time Line

First science important to cloning occurred in

?

1839: Theodor Schwann lays foundation for cell theory

1855: Rudolf Virchow-”All cells arise from cells”

1865: Gregor Mendel-fundamental laws of heredity

1885: August Weismann proposes that genetic information in cells diminishes as cell differentiate during development

1888: Wilhelm Roux’s work supports Weismann

1892: Hans Driesch shows that each cell of a 2-cell or 4-cell sea urchin embryo can develop into a perfectly formed embryo

1894: 1st nuclear transfer experiment by Jacques Loeb

1914: Hans Spemann performs nuclear transfer experiments with newts and in 1928 with salamanders

1938: Spemann asks what would result if a nucleus from a differentiated cell were inserted into an enucleated egg cell

1952: Robert Briggs and Thomas King report development of normal Rana pipiens tadpoles by transferring nuclei from embryonic cells to enucleated egg cells

1953: Watson and Crick report structure of DNA

1962-75: John Gurdon reports cloning frogs using donor nuclei from fully differentiated adult intestinal cells

1979: Steen Willadsen uses single cells from 8-cell sheep & cattle embryos to raise adult animals

1986: Willadsen clones a sheep from embryo cells using nuclear transfer

1990: Human Genome Project officially begins

1995: Ian Wilmot and Keith Campbell use differentiated sheep embryo cells to clone Megan and Morag, the world’s first sheep cloned from differentiated cells

1997: Wilmot and colleagues report the cloning of Dolly, the first animal to be cloned from adult cells

1997: Polly is born at the Roslin Institute-she is cloned from a fetal fibroblast into which the gene for clotting factor IX had been inserted

1998: James Thomson and colleagues report deriving human pluripotent stem cell lines from blastocysts

1998: John Gearhart and colleagues report the derivation of human embryonic germ cell lines from aborted human fetal material

HowHow

is cloning done?

Somatic Cell Nuclear TransferSomatic Cell Nuclear Transfer

• 10%

• 25%

• 33%

• >50%

How successful is cloning?How successful is cloning?

Primates: 1 success in >300 attempts

Megan and Morag were only surviving fertile sheep from 244 nuclear transfers

Dolly was sole survivor from 277 transfers of adult nuclei

In experiments with cattle, goats, and mice success rate is seldom greater than 3% & many abnormalities observed

Idaho Gem, 1 of 3 from 305 transplants

Success RatesSuccess Rates

Normal Looking = Normal Clone?Normal Looking = Normal Clone?

Reported in Science, July 6, 2001

Scientists made mouse clones from ES cellsMonitored activity of imprinted genes

Found that ES cells are unstable in cultureClones made from sister stem cells had differences in gene expression

Imprinted genes important in fetal development so results are a concern for reproductive cloning

The Big QuestionThe Big Question

1. The Time Machine Problem

2. Imprinting

3. Physical damage

4. Extra or Missing donor molecules

5. Lack of understanding of process

Possible ReasonsPossible Reasons

Nuclear Transplantation is Nuclear Transplantation is an Epigenetic Probleman Epigenetic Problem

K. Hochedlinger et al., Reprogramming of a melanoma genome by nuclear transplantation, Genes & Development 18:1875-1885, August 1, 2004.

Embryonic stem cell line created from melanoma

Injecting ES cells into blastocysts produced chimeric mice

ES cells contributed to multiple organs, including skin, heart, and liver

Oocyte cytoplasm reprogrammed melanoma nuclei yielding pluripotent embryonic stem cells

is an “environmental”

issue.

CloningCloning

are born to two different mothers in two different environments in utero,

develop from different eggs & thus have different mitochondrial genomes, and

experience mutations at different rates and at different places in the genome.

Mutations present in the donor’s genome are passed to the clone, and

Clone may start life with telomeres of shorter length than did donor

Clone and donorClone and donor

STEM CELLSSTEM CELLS Embryo

Cloning Human EmbryosCloning Human Embryos

ScienceScience, Vol 303, 1669-1674, 12 March 2004, Vol 303, 1669-1674, 12 March 2004Hwang et al., Seoul National UniversityHwang et al., Seoul National University

Used 242 eggs from 16 women

Donor DNA from cumulus cell of egg donor

Produced 30 embryos

Derived pluripotent embryonic stem cell line from cloned blastocyst

Cell line genetically identical to donor and stable after 70 passages

Imprinting and other analyses confirm embryos derived from donor and did not arise by parthenogenetic activation

Teratomas (containing tissue representative of all three germ layers) formed by human SCNT ES cells in the testes of SCID mice at 12 weeks after injection. Neuroepithelial rosset (A), pigmented retinal epithelium (B), ostoid island showing bony differentiation (C), cartilage (D), and glandular epithelium with smooth muscle and connective tissues (E).

There are ethical, legal, There are ethical, legal, and social issues that and social issues that

must be dealt with.must be dealt with.

Is it important to evaluate Is it important to evaluate separately the issues separately the issues surrounding human surrounding human cloning and those of cloning and those of

animal cloning?animal cloning?

Is concern for Is concern for continuity of species continuity of species or fitness of single or fitness of single

individual?individual?

How does one manage the potential dangers for prospective clones when one is attempting to protect a future potential person

against harms that might be inflicted by their very existence?

How similar (or How similar (or different) are clone different) are clone

and donor?and donor?

Is it appropriate for a Is it appropriate for a person to be his/her person to be his/her

own parent?own parent?

Does cloning Does cloning undermine the undermine the

freedom of human freedom of human beings to construct beings to construct

his/her own life?his/her own life?

Does cloning Does cloning undermine the value undermine the value or worth of human or worth of human

beings?beings?