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The Cloning of Dolly and other Mammals
October 27, 2009



I. What is meant by the term 'cloning'?

  • Greek root =- klon for 'twin' or 'breaking in two'
  • Computers or electronic devices = 'clones'.
  • In science fiction books and movies, 'clones' = replicated copies of an individual, usually resulting in some sort of 'slave army' working for an evil genius, or an evil scientist.... ; )
  • In science = organisms that reproduced by asexual reproduction (like bacteria)
  • Biotechnology = 'cloned genes' - making multiple, identical copies of a gene (or protein) in bacteria.
  • In 1996, 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 (SCNT)....

II. Hello Dolly: Cloning Mammals...
For each cloned animal, list the 'step forward' in cloning progress and research...

 

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 PPL Therapeutics/The Roslin Institute in Scotland quietly announce the birth of Dolly in February, 1997 in the journal Nature, after which, all hell breaks loose...

 

Full Text: Viable offspring derived from fetal and adult mammalian cell [PDF] Nature 1997 Feb. 27; 385(6619) Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH Roslin Institute (Edinburgh), Roslin, Midlothian, UK. [Image]

 

 

 

 

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

More detail on those Early Steps: Images from Scientific American, November 2000; another nice fig. in Human Molecular Genetics 2
Only 3 ingredients needed!

  • 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...
  • 1 Surrogate mother!

Images from Time Magazine, March 10, 1997:

 



 

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. Timeline: Since then, much work has been done to streamline and refine the cloning process in laboratory animals, livestock, and even pets:

 


1997: Hello Polly The first sheep cloned by nuclear transfer technology bearing a human gene (Factor VIII), by PPL Therapeutics / Roslin Institute.
April 1998: Dolly gives birth to Bonnie: conceived in the good old-fashioned way. Triplets the year after.

 



October 1997:Cumulina: Three Cloned Mice: 22 of her cloned siblings (some cloned from clones) using the 'Honolulu Technique'. Nature 394:369-374. Author Teruhiko Wakayama concludes that "contrary to previous opinion, mammals can be reproducibly cloned from adult somatic cells"



April 1999: First (three) cloned goats; Mira, Mira, and Mira..., Louisiana State University and Tufts University School of Veterinary Medicine, Nature Biotechnology.

 

Dec. 1999: Mice cloned from ES cells PNAS 96:14984. Teruhiko Wakayama again - "ES cell nuclei can be used to produce viable cloned mice and provide a link between 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."



 

May 5 1999: Roslin Bio-Med becomes Geron Bio-Med.

 

August 2000: First cloned Piggies:Roslin Institute / Geron and National Institute of Animal Industry in Japan (cute picture!) simultaneously publish in Science and Nature

 

August 2002: Cloned Pigs Raise Transplant Hopes (see below)

February 2002:First cloned kitty - Cc and surrogate mother Rainbow.

14 Feb. 2003:"Dolly the sheep, the first cloned mammal, is dead at age 6, of a lung infection. Her death probably had nothing to do with the fact that she was a clone, said Dr. Ian Wilmut. "The fact the she isn't there any more will be brought home to us when we go near that barn and she's not there," Dr. Wilmut said. Snif.

16 December 2006: World's first cloned cat has 3 healthy kittens Copy Cat became a mother the old-fashioned way, says scientist

 


29 May 2003:Way to go, Idaho!
: A healthy mule named Idaho Gem is the first member of the horse family to be cloned. Yee-hah!

25 May 2006: 'Its Idaho Gem ahead of Idaho Star by 0.03 sec'! These two are brothers, cloned from cells of Taz, a champion mule.

7 August 2003: World's first cloned horse born to its genetic twin: -- "Italian scientists have created the world's first cloned horse, Prometea, from an adult cell taken from the horse who gave birth to her. ". Nature 2003 Aug 7;424:635. Pregnancy: a cloned horse born to its dam twin.


26 Sept 2003: Rat joins list of cloned animals The French announce a new twist on cloning - rats (great models for diabetes and hypertension) have been much more difficult to clone because their oocytes (necessary for SCNT/cloning) speed through meiosis. Zhou et al succeeding in cloning Ralph (the rat) by using a drug to block the first metaphase-anaphase transition, a technique they call delayed oocyte activation.

 

Published in Science 26 September 2003 Vol. 301 Generation of Fertile Cloned Rats by Regulating Oocyte Activation

22 April 2004: Mouse created without father Kaguya "(Japanese folk tale) Mouse born by combining 2 egg nuclei. Kaguya was the ONLY pup of 460 attempts (but healthy and fertile). Key to the success= use of an immature, female egg that lacked key imprinting proteins H19 and Igf2, making the egg more sperm-like". Birth of parthenogenetic mice that can develop to adulthood Nature Tomohiuro Kono et al., 428, 860 - 864 (Classic understatement: "we have shown for the first time that it is possible to obtain a viable adult mouse from two maternal genomes."). See also
Mickey Has Two Mommies 20 August 2007.
Twenty-seven "bimaternal" mice survived to adulthood!!!


5 August 2004: Tabouli and Baba Ganoush The Genetic Savings and Clone - Two kittens have been born using a new cloning method (chromatin transfer) that may be safer and more efficient than traditional methods,

11 October 2006: Too bad: Genetic Savings & Clone to Close its Doors...not a big enough market for $32,000 kitties! >^.^<


3 August 2005: Announcing Snuppy:
The first cloned puppy, "Seoul National University Puppy" The work was completed by the lab of Woo Suk Hwang, the discredited South Korean researcher who is (in) famous for publishing falsified data claiming that his research team had derived patient-specific stem cells.

And I think one of the best cloning papers of ALL TIMES:

Cloned ferrets produced by somatic cell nuclear transfer Li and Engelhardt, Dev Biol. 2006 May 15; 293(2): 439–448. One of my favorite papers of all times for a good, clean set of experiements, way cute baby cloned ferrets Libby and Lilly, and a nice readable style that generates a few good laughs...:)

 


Cloning Ferrets = WHY?!? The domestic ferret (Mustela putorius furo) :)

= ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis

  • 'Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies.
  • Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential and delayed Oct4 expression in recipient oocytes .
  • Oocyte stimulation was instead induced by mating to a vasectomized male.
  • TWO methods for SCNT = Nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (~3–4%), only cell fusion gave rise to healthy surviving clones.
  • With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases.
  • Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.'

V. Why Clone Mammals?

1. Cloning of Livestock: For the genetic propagation of animals that are especially high producers of a desired product, allowing for the production of herds that can be farmed (or "pharmed") for milk, blood and organs: [Image] Uses: * Human therapeutic proteins * Organs and tissues for transplants * 'Humanized' cows milk * Animal models of disease * Cell therapy agents

The big livestock cloners:
The Roslin Institute Edenburgh, Scotland
Advanced Cell Technology Worcester, MA
Cyagra, Elizabethtown, PA

Three recent developments in Cloned livestock:

(1) DKO Piggies for organ transplants: Christmas Day, 2001:(Press release) Race for the piggies: Knockout pigs designed for human transplants =lack one allele for production of a sugar, alpha 1,3 galactose that causes a rapid 'hyperacute rejection' of pig organs transplanted into humans. The University of Missouri-Colombia and Immerge BioTherapeutics Inc, however, published results of their litter of knockout pigs almost simultaneously in Science 295:1089-1092. Production of -1,3-Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning

 

August 22, 2002: It's a first! Double knockout pig: The Roslin Institute = piglets that are double knockouts for alpha 1,3 galactose "Because both copies of the gene have been inactivated, tissues from these pigs are completely devoid of the pig sugar that cause the hyperacute rejection to take place.", Cloned Pigs Raise Transplant Hopes


(2) TC Calves with human immune system August 12, 2002: Human immune system in cloned calves. " Hematech (SD) and Japanese brewing company Kirin announce the birth of 4 healthy trans-chromosomic, cloned calves (TC calves) producing human antibodies in the journal Nature Biotechnology. The team inserted a human artificial chromosome into cloned cow embryos. The chromosome carried the genes for two proteins that make human antibodies.

 

"The first step in the project, which we have accomplished, is to put the human immune system genes into cows. The second step, which is in progress, is to remove the cow immune system genes. Once both steps are completed we will be ready for production. This gives us the ability to produce a wide variety of complex, natural therapeutics (antibodies) that will help people to fight many different kinds of disease." Nat Biotechnol. 2002 Sep;20(9):889-94 Cloned transchromosomic calves producing human immunoglobulin.


(3) a-1-antitrypsin sheep for treatment of emphysema and other respiratory disorders. PPL /Bayer to produce this drug that had indications for treating a wide variety of respiratory disorders - CF, emphysema, chronic obstructive pulmonary disease and others characterized by an excess of neutrophil elastase. Sadly, clinical trials indicated that the drug did not perform as expected, and produced a mysterious cough in recipients of the drug, leading Bayer to stop clinical trials in June, 2003 . The fate of the thousands of sheep in the a-1-antitrypsin herd hung in the balance...but the protein may turn out to be useful as a surgical sealant, so there may be some use for this product yet! Update: PPL Therapeutics was acquired by The University of Pittsburgh Medical Center and sold its nuclear transfer patents to Exeter Life Sciences of the US 01-Jan-2004

 

Plus,

4) I'll have the TC Calf-burger with a side of Bt-fries, please...ooh yummy.
September 2, 2002 Is is safe to eat cloned livestock? The National academy of Sciences says Yes; but the FDA says to wait a bit until safety issues can be examined more closely.

 

28 December 2006:

FDA Issues Draft Documents on the Safety of Animal Clones
Agency Continues to Ask Producers and Breeders Not to Introduce Food from Clones into Food Supply. "Based on FDA's analysis of hundreds of peer-reviewed publications and other studies on the health and food composition of clones and their offspring, the draft risk assessment has determined that meat and milk from clones and their offspring are as safe as food we eat every day," said Stephen F. Sundlof, D.V.M., Ph.D., director of FDA's Center for Veterinary Medicine.

"Cloning poses no unique risks to animal health currently in use in U.S. agriculture."

BUT, "Because the release of the draft risk management plan marks the beginning of our interaction with the public on these issues, we are continuing to ask producers of clones and livestock breeders to voluntarily refrain from introducing food products from these animals into commerce so that we will have the opportunity to consider the public's comments and to issue any final documents as warranted," said Sundlof.

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, a Cow-Guar hybrid. (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...). For more info about cloning endangered species, please read this Good For. [Cloning your deceased pet also falls into this category...]

 

Endangered species to clone? Extinct species?
Hmm, can we think of any OTHER MAMMALS we might want to clone? I wonder.....

 


VI. Human Reproductive Cloning?!?


Cloning primates...still not there yet!

The Problem With Cloning Primates 10 April 2003- "While governments debate how to prevent human reproductive cloning, it seems that nature has put a few hurdles of its own in the way. Simerly et al. report that in rhesus monkeys, cloning robs an embryo of key mitotic spindle proteins, called NuMA and HSET, that allow a cell to divvy up chromosomes and divide properly. The same problem may also thwart attempts to clone humans." Science, Vol 300, Issue 5617, 297 Molecular Correlates of Primate Nuclear Transfer Failures

Time Magazine, February 2001 Human Cloning is closer than you think
Time Magazine, August 2001 Is Human Cloning an Inevitability?

 

August 7th, 2001: Human cloning conference: a joint panel of the National Academy of Science Committees on Science, Engineering Public Policy and the Board of Life Sciences = to discuss the ethical and scientific concerns of human cloning; however,

 

"Despite warnings from scientists present, 3 privately-funded US / European groups vowed at the meeting (and on televised coverage of the meetings) to continue their programs on Human Cloning." First human clone bid planned:

 

 

The three groups currently attempting to clone humans for reproductive purposes:

1. Dr. Severino Antinori: of Italy, who became infamous in 1994 when he helped Rosanna Della Corte, a 63-year-old woman give birth.    Plans to use the man's genetic material and the woman's egg (and egg cytoplasm, which contains the woman's mitochondria).

2. Dr. Panos Zavos: of Lexington KY, director of the Andrology Institute of America, and the Kentucky Center for Reproductive Medicine.

3. Dr. Brigitte Boisselier: scientific director of Clonaid, "The First Human Cloning Company" . Founded several years ago by Rael, a spiritual leader of the Raelian Movement, who believes that human life is the result of extraterrestrial genetic experiments.

December 27, 2002: The Raelians announce by press release (on a very quiet news day) Baby Eve, 'the first cloned baby': Is this the first step towards human immortality? OR, maybe just a media circus.... Without scientific data, we'll never know...

  • "Nobody should get a story until they produce evidence. No matter how telegenic they are, no matter how many Star Fleet Command uniforms they have, if you don’t show up with a baby or a parent or a DNA test, or some witnesses who are credible, you shouldn't’t have a story." We're still waiting!
    -- Arthur Caplan Director, Center for Bioethics, University of Pennsylvania
  • Feb 2003 Nature Biotechnology: Generating Copies: Scary thought: "A secretive group like Clonaid might be the perfect organization for carrying out a reproductive cloning program. The main requirements would be:
      1. a small team of researchers willing to carry out the work, irrespective of the appalling risks to the mother and child,
      2. an initial outlay of equipment necessary for IVF,
      3. lots and lots of human eggs...."
  • March 2003 Scientific American: The Rael Thing. Funny...!
  • April 2003: DNA pioneer urges gene free-for-all YIKES...What was James Watson thinking? Has he been taken aboard the mother ship? Sometimes, Nobel Laureate or not, its best to keep your mouth SHUT!

Moral: Human Reproductive cloning to make babies? NO!!! Cloning mammals is currently so inefficient, and so likely to result in gestational or neonatal developmental abnormalities, it is currently outside the realm of acceptable science / medicine. Let's stop talking about it!


VIII. Safety Issues:

Could it be said any better than this? Don't * Clone * Humans! Rudolf Jaenisch and Ian Wilmut Science. 2001 Mar 30: (Numbered points from their article):


1. Animal cloning is inefficient and is likely to remain so for the foreseeable future.

  • It took 277 attempts to get 1 cloned sheep - Dolly.
  • It took 188 attempts to get 'Cc'.
  • It took 305 attempts to get Idaho Gem and Utah Pioneer
  • It took 460 attempts to get Kaguya
  • Typically hundreds of attempts are made before one 'successful' clone is born alive and well.

2. Cloning results in gestational or neonatal developmental failures

  • At best, a few percent (<4%) of the nuclear transfer embryos survive to birth and, of those, many die within the perinatal period.
  • Many spontaneously miscarry or the die shortly after birth.
  • Placental malfunction = embryonic death during gestation.
  • Respiratory distress and circulatory problems = most common causes of neonatal death
  • Even apparently healthy survivors may suffer from immune dysfunction, or lung, liver, kidney or brain malformation.
  • Many survivors are oversized = "Large Offspring Syndrome" Gene Find No Small Fetus Wired, Jan 2001 (Large Offspring Syndrome and Igf2R expression)
  • But: Scientists at ACT do have evidence: Cloned Cattle Can Be Healthy and Normal Lanza et al. Science, Vol 294, Issue 5548, 1893-1894 , 30 November 2001

3. The most likely explanation for the low rate of success may be failures in epigenetic reprogramming and imprinting - mechanisms to ensure that a zygote ends up with a 'clean slate' from two different parents, and that the 'interests' of each parent are balanced.

  • From "Evolution of imprinting mechanisms: the battle of the sexes begins in the zygote" Nature Genetics 27: 255 - 256 "Imprinting = differential gene expression depending upon parent-of-origin of allele = one is repressed, one is transcribed. There are over 40 imprinted genes known in mammals (Table) commonly silenced by methylation or siRNA (Figure)". Most imprinted genes are involved in fetal, placental and neonatal growth, with "paternally derived genes tending to foster large offspring, while maternally derived genes would moderate growth to safeguard the mother", according to the so-called "Genetic conflict" hypothesis.
  • "Imprinting is normally accomplished during spermatogenesis and oogenesis, processes that in humans take months and years, respectively. During SCNT, reprogramming of the somatic donor nucleus must occur within minutes or, at most, hours between the time that nuclear transfer is completed and the onset of cleavage of the activated egg begins. It is known that the cytoplasm of an oocyte can reprogram the genome of a somatic cell to an embryonic state, but the exact mechanisms are not clear.
  • Epigenetic Reprogrammming is a normal process in gametes and early embryo (and must also happen in SCNTembryos) that re-sets the correct embryonic pattern of gene expression. As cells begin to differentiate, cell-type-specific patterns of DNA methylation and histone modifications are acquired"

 

Changes in imprinting are seen when examining different clones of the same organism, suggesting again that subtle changes in gene expression occur during the cloning process.

Final thought from Rudolf Jaenisch and Ian Wilmut

Humans not fit for cloning, Rudolf Jaenisch, MIT Biology Prof. Wired, Sept 2002 (general health problems with cloning)
"
Don't * Clone * Humans! There is no reason to believe that the outcomes of attempted human cloning will be any different...."

 


Objectives:

1. Please be able to state 'who' these cloned animals are and why we are discussing them: Dolly, Polly, Cumulina, Noah, Cc, Ihaho Gem, Ralph, Kaguya, Prometea, and Snuppy.
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, and specific examples like DKO pigs and TC calves.
6. List two reasons scientists are interested in cloning animals like guars, and like cats and dogs.

7. Explain how imprinting and epigenetic reprogramming may be involved in developmental errors in cloned animals
8. What is human Reproductive Cloning? Who is pursuing this procedure?

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