The Cloning of Dolly, 'Cc', and other Mammals
February 25 and Feb 27, 2002

Note: This is a long one..but we will take two days to discuss the material
Optional Readings: Announcing Dolly: Scientific American, March 1997
The Roslin Institute - Information on Cloning (Dolly's birthplace in Bonny Scotland)
Cloning Noah's Ark Scientific American, November 2000
Slouching Towards Creation (A weird but fun site; don't look at it late at night)

I. What is meant by the term 'cloning'? Cloning refers to making an exact copy of an original form.

• The root of the word comes from the greek word for 'twin' or 'breaking in two' - klon.
• Computers or electronic devices that look like an original are said to be 'clones'.
• In science fiction books and movies, 'clones' implied replicated copies of an individual, usually resulting in some sort of 'slave army' working for an evil genius, or an evil scientist.... ; ) See this week's Warm Up to cast a vote for the cloning academy awards! !
• In science, the term was originally used to refer to organisms that reproduced by asexual reproduction (like bacteria)
• Once biotechnology took off in the 1980s, scientists learned how to splice genes from humans or other organisms into bacteria and started to refer to 'cloned genes' - genes placed into bacteria to be replicated - 'cloning' simply meant making multiple, identical copies of a gene (or protein) in bacteria.
• In 1996, cloning took on a new meaning: an new individual grown from a single somatic cell of its parent and genetically identical to it... This technique is also called somatic cell nuclear transfer....

II. Hello Dolly: Cloning Mammals...

July 5, 1996: First mammal cloned from adult cells: A surrogate mother sheep gives birth to Dolly, a lamb cloned from an udder cell of an adult sheep born 6 years earlier. Ian Wilmut and colleagues at the PPL Theraputics and the Roslin Institute in Scotland quietly announce the birth of Dolly in February, 1997 in the journal Nature (Viable offspring derived from fetal and adult mammalian cells. Nature 1997 Feb 27; 385(6619) Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH Roslin Institute (Edinburgh), Roslin, Midlothian, UK.)

Dolly is a later-born genetic twin of the somatic cell she was cloned from(a female Finn Dorset Ewe).

Since then, much work has been done to streamline and refine the cloning process in livestock and in laboratory animals like mouse:

• Hello Polly (1997) The first sheep cloned by nuclear transfer technology bearing a human gene (Factor VIII), by PPL Theraputics / Roslin Institute.
• 'Gene' the Bull - (August 1997) cloned by ABS Global. (but cloned from fetal, not adult, cells)
• Cumulina: (October 1997) Three Cloned Mice: Dolly is joined on October 3, 1997 by the cloned mouse "Cumulina" and, shortly afterward, by 22 of her cloned siblings (some of whom were cloned from clones) using the 'Honolulu Technique' of nuclear transfer. Author Teruhiko Wakayama concludes that "contrary to previous opinion, mammals can be reproducibly cloned from adult somatic cells" [Image]
• Dolly gives birth to Bonnie: (April 1998) conceived in the good old-fashined way. (ie: Dolly was healthy and fertile). Triplets the year after.
• Dolly's telomeres are shorter than other 3-year-old sheep, (May 27 1999 Nature) but Dolly seems to be just fine, maybe just a bit more obese that other sheep her age...
• 1999, Roslin Bio-Med becomes Geron Bio-Med. Geron buys part of the Roslin Institute. Gotta love their patent position: telomerase, hES cells, AND somatic cell nuclear transfer!
• Mice cloned from ES cells (December 1999), Teruhiko Wakayama and colleagues are at it again - "Our findings demonstrate that ES cell nuclei can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period."

III. Somatic Cell Nuclear Transfer: (aka cloning). A how-to:

Ingredients:

• 1 somatic (diploid) donor cell, containing the complete genome of that individual +
• 1 unfertilized recipient egg with its haploid nucleus removed (or 'e-nucleated') +
• 1 electric shock to get them together...

Images from Time Magazine, March 10, 1997

More detail on those Early Steps: Scientific American, November 2000
Quiescence: The state in which all but the most basic functions of a cell or group of cells has stopped. This is usually a response to an unfavourable environment, such as one in which the food supply is low or absent. The cell becomes dormant until its surroundings are more favourable. In this state the genes that define the specialist function of a cell "switch off" making the cell suitable for nuclear transfer.

IV. Why do scientists want to clone animals?!?!?

1. The Potential of cloned livestock: (September 1997) Scientific American. Cloning livestock allow for the genetic replication of animals that are expecially high producers of a desired product, allowing for the creation of animal herds that can be farmed for milk, blood and organs: [Image]

* Human therapeutic proteins (hormomes, clotting factors, etc)
* Organs and tissues for transplants
* 'Humanized' cows milk
* Animal models of disease (diabetes, muscualr dystrophy, etc)

The big livetock cloners:
Advanced Cell Technology
ABS Global
The Roslin Institute

2. A second reason: Saving endangered species: Cloning Noah's Ark (November 2000) Scientific American. The inspiring (but ultimately sad) story of the cloning of the baby Guar, Noah. See this week's Good For. (Even sadder than NOT being able to clone endangered species? - the daily extinction of species due to the continual, daily habitat destruction for numerous species worldwide....thanks to humans...)

3. A third reason: Cloning your deceased Pet?!?!? Lassie, come home.

• Bank your pet's DNA at the Genetic Savings & Clone
• The Missyplicity Project is a project funded by the Genetic Savings & Clone to produce the first cloned dog. They have invested $2.3 million to produce a clone of a beloved pet dog, Missy, a mixed breed border collie. A team of about 20 researchers is working on the Missyplicity Project.
• 2/14/02 Operation Copycat First Cloned Cat is born. "Texas A&M announced today the first cloned kitten, appropriately named Cc. 'She is as cute as a button,' said a spokeswoman for Texas A&M, where the work was done using a grant from philanthropist John Sperling's Genetic Savings & Clone. 'The kitten was vigorous at birth and appears to be completely normal,' Mark Westhusin and colleagues write in their report in a letter published in the science journal Nature.
• The kitten is a calico-and-white shorthair that looks similar to, but not exactly like, her genetic mother. The scientists said her coat coloring was unique because not only genetics contribute to an animal's markings, but also conditions in the womb.
• Cc, like cumulina, was cloned from a cumulus cell. These cells nurture the developing eggs in a female's ovary and have been used to clone other animals as well, as they seem particularly adaptable to the process." [Image and quotes]
• Wondering what to give that special someone who has everything?? Why not a gift certificate good at any Genetic Savings and Clone?

V. Cloning Human Beings...? (Will there ever be another 'ewe'?)

"There is no clinical reason why you would clone humans. Why would you make another human being? We think it would be ethically unacceptable and certainly would not want to be involved in that project.'' Dr. Ian Wilmut, The Roslin Institute

"We will get there, because very simply it's a matter of determination. And I think we are determined to get there," Dr. Panos Zavos, University of Kentucky, who, beginning in November 2001, will attempt to clone the first human, with the help of over 200 volunteer infertile couples. (Check back in 9 months..)

One reason to not pursue human cloning:

July 16 2001: H.R. 2505, Human Cloning Prohibition Act of 2001, Introduced by Rep Dave Weldon. Passed by a vote of 265 to 162 in the US House of Representatives a ban on all human cloning, either for reproduction or for therapeutic cloning. The bill, if passed into law, would make cloning a crime punishable by up to 10 years in prison and outlaw the sale of any treatments developed from cloning. Currently faces uncertain prospects on the Senate Calendar, particularly after September 11 (other things to worry about...). Note: Two dozen countries ban human cloning, but there is no such cloning ban in England and many other countries...and really none now in our country...until this act works its way through Congress and the White House.

Two reasons some scientists and physicians want to pursue human cloning:

1. Reproductive cloning: making babies for infertile couples created from a single somatic cell without sexual reproduction.

2. Therapeutic cloning: making immunologically compatible hES cells (tissues and maybe someday organs...) for a person who has disease, a disorder, or an injury (diabetes, leukemia, spinal cord injury, etc. etc.). Some scientists advocate calling this technique "nuclear transplantation" to avoid the negative connotations of cloning.

VI. Future Games: Reproductive Cloning:

Time Magazine, February 2001 Human Cloning is closer than you think
Time Magazine, August 2001 Is Cloning an Inevitability? (Time: moral compass)

First human clone bid planned: Despite warnings, on August 7th, 2001, at a National Academy of Sciences meeting, 3 privately-funded US / European groups vow to contunue their programs on Human Cloning.

Dr. Severino Antinori: of Italy, who became well-known in 1994 when he helped a 63-year-old woman to have a baby by implanting a donor's fertilised egg in her uterus, making her the oldest known women in the world to give birth.    Plans to clone babies starting November 2001! - for couples who are infertile because the man is unable to make sperm, using the man's genetic material and the woman's egg (and egg cytoplasm, which contains mitochondria).

Dr. Panos Zavos: of Lexington KY, director of the Andrology Institute of America, and the Kentucky Center for Reproductive Medicine. Would like to clone humans by 2003, collaborating with Dr. Antinori to impregnate up to 200 women from infertile couples with cloned embryos in hopes that at least a few of the women will carry a child to term.. (Note from Dr. Marrs: In the immortal words of Buzz Lightyear, from Toy Story (1995), "I don't believe that man has ever been to medical school".)

Dr. Brigitte Boisselier: scientific director of Clonaid, "The First Human Cloning Company". Founded three years ago by Rael, a spiritual leader of the Raelian Movement, who believes that human life is the result of extraterrestrial genetic experiments. (Note from Dr. Marrs: In the immortal words of Fozzie Bear, from The Muppet Movie (1979), "We picked up a weirdo...!")

Scientist's responses: not good. "Practice, it is said, makes perfect. But is it ethical to practice? And I absolutely think it is not, in the human context," said Alan Colman, a researcher for Scotland's PPL Therapeutics.

Jeremy Rifkin, do you have anything to add? (Jeremy Rifkin is a vocal opponent of the Biotechnology industry)

VII. Therapeutic Cloning:

Teruhiko Wakayama cloned mice from ES cells and other labs have done this with livestock. This provides a link between the technologies of ES cells and animal cloning.

Therapeutic cloning involves the creation of an embryo via somatic cell nuclear transfer (cloning) from an animal (ie: a person) with a disease, to derive hES cells immunologically identical to donor, in hopes of treating the disease. The cloned embryo would exist only until day 5, when it would be used to isolate hES cells.

• The nucleus of a somatic cell from the donor (ie: you?) would be removed and placed into an enucleated human egg.
• "Fertilization" would be simulated with electric shock.
• The inner cell mass would be removed from the 5-day old blastocyst, which is genetically identical to the donor.
• hES cells would be cultured into the tissue type needed by the donor.
• This immunologically identical tissue would be placed back into the donor, hopefully curing the donor's disease

Eventually, growing replacement organs (heart, liver, pancreas, skin, etc) MAY be possible - however I think we can conservatively say we are many years away from producing needed tissues, let alone whole organs.

A step forward...or back? Thanksgiving weekend, 2001:

• Sunday, November 25, 2001: The First Cloned Human Embryo "ACT scientists reported that they had used somatic cell nuclear transplantation to create several human embryos. Unfortunately, only one of the embryos progressed to a 6-cell stage, at which point it stopped dividing and died (note from KM: the numbers go: 1 cell, 2 cells, 4 cells, 8 cells....obviously something was wrong there!) and none were able to get to the 5-day blastocyst stage. E-biomed Rapid Communication: Somatic Cell Nuclear Transfer in Humans: Pronuclear and Early Embryonic Development the Journal of Regenerative Medicine  2 (25 - 31) Jose B. Cibelli; Ann A. Kiessling; Kerrianne Cunniff; Charlotte Richards; Robert P. Lanza; Michael D. West
  
• Monday, November 26, 2001: Scientific criticism: Experts rip cloning 'story' "Glenn McGee, a University of Pennsylvania bioethicist resigned today from Advanced Cell Technology's ethics advisory board, calling the announcement 'nothing but hype. They are doing science by press release,' he said." Scientific criticism from every direction directed at ACT, Scientific American, and the Journal of Regenerative Medicinebecause it was not good science.
  
• Tuesday, November 27, 2001: Bush denounces ACT WASHINGTON- AP: "President Bush, joined by a chorus of religious and political leaders in the United States and abroad, today condemned the work of a Massachusetts biotechnology company that says it has created human embryos through cloning. The White House called on lawmakers to ban the practice. 'The use of embryos to clone is wrong,' Mr. Bush said this morning at a Rose Garden ceremony 'We should not as a society grow life to destroy it.'
  
  "The president's comments, coming on the heels of the company's announcement on Sunday, reignite an intense debate that had been all but extinguished by the Sept. 11 attacks. The Senate is facing increased pressure to consider legislation, similar to a bill passed by the House of Representatives in July, that would prohibit human cloning either for reproduction or to create tissue for treating disease. Hours after Mr. Bush spoke, Senator Sam Brownback, Republican of Kansas, said he would push the Senate to adopt the bill before its Christmas recess. If lawmakers refuse to do so, Mr. Brownback said, he will press for a six-month moratorium that would temporarily ban the cloning research." [Quotes and image from the New York TImes]

IV. Safety Issues:

Cloned Cows Dropping Like Flies April 2, 2001
Study Raises Human Cloning Doubts July 6, 2001
Imprinting marks clones for death. Nature, July 2001
Clone Snafu suspected Wired, May 2001

The rate of success of cloning is currently very low - cloned animals rarely make it to adulthood - or sometimes even birth.

• It took 277 attempts (failed pregnancies) to get 1 cloned sheep - Dolly.
• It took 188 tries (failed pregnancies) to get just one 'Cc'.
• Some labs are better at it than others, but typically a hundred or so attempts are made before one 'successful' clone is born alive and well.
• Many times, the placenta of the animal is abnormal, contributing to a miscarriage.
• Many animal embryos spontaneously miscarry or the animal dies shortly after birth.
• Cloned animals often have problems with their immune systems, lungs, and livers, causing death at an early age.
• They also have problems with obesity.

A recent study in Science and another in Nature in which researchers monitored gene expression in embryo and fetal development showed that mice cloned from ES cells had subtle changes in many of the genes involved in early development of the embryo and of the mouse after birth - changes that may not cause noticible defects in physiology or behavior, but may alter individuals slightly.

Often genes are 'imprinted' during development - turned on or off depending on whether they came from the maternal or paternal parent. Changes in imprinting are seen when examining different clones of an organisms, suggesting again that subtle changes in gene expression occur during the cloning process.

Although telomeres of cloned animals can be shorter than their non-cloned relatives, the significance of this finding is not yet known.

PS. Happy Birthday to ewe, Happy Birthday to ewe...Dolly turned 5 (or is it 11?) on July 5, 2001. The lifespan of a sheep is about 6 years.

V. Ethical Issues (we will do some worksheets about this both Monday and Wednesday):

What are the benefits of and who benefits from reproductive cloning?
What are the potential drawbacks or harmful things that could result from reproductive cloning?
Imagine a child growing up as a 'clone' of an adult from a member of her family. What are some of the 'issues' she might have to deal with (besides the usual issues like "nobody understands me" and "why can't I get my ears pierced?")

What are the benefits of and who benefits from therapeutic cloning?
What are the potential drawbacks or harmful things that could result from therapeutic cloning?

Would you be willing to have rules or decisions about human cloning applied to you or your family members?

Read if you are interested:

The Vatican: Reflections on Cloning
21 arguments against human cloning and their responses

Objectives:

1. Please be able to state 'who' these cloned animals are and why we are discussing them: Dolly, Polly, Cumulina, Noah, and Cc.
2. Who is Ian Wilmut? (no, he is not a cloned mammal!)
3. Define cloning as it relates to mammals.
4. Describe the 'ingredients' needed to make a cloned mammal.

• What is the purpose of the diploid nucleus?
• Why is the recipient egg enucleated before the procedure?
• What is left in the recipient egg after e-nucleation and why is this important for the developing animal?
• What is the purpose of the electric shock?
• What is quiescence and why does it appear to be crucial to cloning mammals?

5. List three reasons scientists are interested in cloning livestock animals or research animals like mice.
6. List two reasons scientists are interested in cloning animals like guars, and like cats and dogs.
7. What is the Human Cloning Prohibition Act of 2001? What has prevented this Act from progressing through the Senate and George Bush's desk?
8. What is human Reproductive Cloning? Who is pursuing this procedure?
9. What is human Therapeutic Cloning? Why may this procedure be needed to realize the potential of hES cells?
10. List 4 medical and safety issues concerning cloned animals.