Keys to Science, August 2004

Ditto Gene Neti Cumulina Megan, & Morag Idaho Gem Who are

Dolly & 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 G0G0 & Normal Cloning

? Bottom line Bottom line:

Cloning 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 : 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

How is cloning done?

Somatic Cell Nuclear Transfer

10% 25% 33% >50% 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 Rates

Normal Looking = Normal Clone? Reported in Science, July 6, 2001 Scientists made mouse clones from ES cells Monitored activity of imprinted genes Found that ES cells are unstable in culture Clones 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 Question

1.The Time Machine Problem 2.Imprinting 3.Physical damage 4.Extra or Missing donor molecules 5.Lack of understanding of process Possible Reasons

Nuclear Transplantation is an Epigenetic Problem K. Hochedlinger et al., Reprogramming of a melanoma genome by nuclear transplantation, Genes & Development 18: , August 1,  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. Cloning

 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 donor

STEM CELLS Embryo

Cloning Human Embryos

Science, Vol 303, , 12 March 2004 Hwang 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, and social issues that must be dealt with.

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

Is concern for continuity of species or fitness of single 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 different) are clone and donor?

Is it appropriate for a person to be his/her own parent?

Does cloning undermine the freedom of human beings to construct his/her own life?

Does cloning undermine the value or worth of human beings?