Edgey about BioTecknowledgey: A Case Study of Cloning

Commentary by Tom Zinnen

The story grew. On Monday, February 24, the president of the US gave his ethics advisory board 90 days to produce a report assessing the ethics of the new technology and providing ways to prevent is abuse.

The story peaked and receded on Tuesday and Wednesday--yet the research paper describing Dolly was not available until Thursday, February 27. Before the science could be scrutinzed, the first wave of the story had run its first course. First impressions were pressed without benefit of really knowing what had been done.

The following Monday a new wave rose. Cloning made the cover of Time and Newsweek (both were dated March 10, 1997, but actually were released a week earlier). And the president of the US issued an executive order banning any federal money from being spent on research into cloning humans. And in an unprecedented move, he asked for a moratorium on such research by private individuals and institutions.

In this case study we'll analyze a paper that, for scientific and social impact, will be a unique classic. It is available at www.nature.com, and you will need a paper or electronic copy of the paper to make best use of this case study.

The paper:
Nature, Volume 385, 810 - 813, February 27, 1997
Viable offspring derived from fetal and adult mammalian cells
I. Wilmut, A. E. Schnieke*, J. McWhir, A. J. Kind* & K. H. S. Campbell
Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK
* PPL Therapeutics, Roslin, Midlothian EH25 9PP, UK

Overview:

Overview of what some people thought happened (as of February 25, 1997):

Researchers took a cell from an adult sheep,transferred some DNA, grew it somehow and somewhere (in a test tube, in a surrogate ewe??) and produced a lamb genetically identical to the original adult sheep. Furthermore, this technique will likely work on humans, and so the age of human cloning is upon us.

Analyzing the title.

• "What terms do the authors use in their report?"
  
  Notice the language: "viable offspring" rather than "clone"; "derived from" rather than "cloned from" or "born" or "generated."
  
  
• What cells do the authors use in their work?
  
  Note that both fetal cells and adult cells of sheep were the source of the offspring. Dolly was the only offspring derived from an adult cell's nucleus, however.

The Opening Sentence.

• What terms are used to describe the various cells and processes involved in sexual reproduction and development and cell differentiation and gene expression? Keep in mind the progression of cells and processes: oocytes and spermatocytes undergo meiosis to produce gametes (egg, sperm), cell fusion '(plasmogamy)' and nuclear fusion (karyogamy) leading to a zygote (diploid),which divides by mitosis and cytokinesis to form an embryo, which develops into a fetus, with differentiation of tissues and generation of organs; the fetus is born and the offspring develops into an adult.
  
  The Second Sentence: "Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification."
  
  
• Before February 23, 1997, what was the dominant view or paradigm regarding the possibility of using a cell from an adult mammal to generate a new animal?
  
  "A basic idea of cell biology is that diffentiated cells contain all the genetic information as the zygote from which they were derived." If so, then how to explain that a single zygote cell can divide, differentiate and develop into a fetus, but single cells from adults apparently cannot? The key question: are the genes of cells of adults, cells that have differentiated, changed in some irreversible way that the cells cannot develop into adults?
  
  
• To this question there are two possible answers: the genes of the cells are changed irreversibly, or they are not.
  
  But testing these two possiblities poses a dilemma. This is like asking whether a seed is viable: if you plant it and it grows, you know it was viable. If you plant it and it doesn't, you're don't know if the seed is dead or if it's dormant and you just don't know how to wake it up.
  
  
• In this experiment,there are two possible results: either the cell isolated from an adult does grow into another adult, or it does not.
  
  If it does, the answer is clear--and the implications are resounding: this would disprove the idea of "irreversible genetic modifications" in adult cells.
  
  If it does not, there are two possible explanations: the cell didn't because it can't; or the cell didn't because it was not cultivated under the appropriate conditions.
  
  Technical Ingenuities: Nuclear Swapping Through Cell Fusion, and Using Quiescent Cells.
  
  
• In the case of Dolly, did the researchers use only one cell isolated from an adult? Or did they actually build a combination cell from two cells?
  
  In fact, although the authors use the term "nuclear transfer" in the methods section it becomes clear that they fused two cells together. One cell had a nucleus, and came from a population of cells grown "in vitro" (in test tubes) after originally being taken from the udder of an adult sheep. The second cell was an unfertilized egg cell from which the researchers had removed the nucleus. While the researchers called it "nuclear transfer" and some wags called it "nuclear swapping", could it not also be considered "cytoplasmic swapping"? Could there be something in the cytoplasm of the egg cell that activates the genes of the nucleus from the cell derived from the cells from the udder?
  
  
• The authors point out they intentionally used "quiescent" cells. What are quiescent cells? How are they made? How would you vigorously test the hunch or speculation that quiescence is significant and that quiescence synchronizes the two cells?
  
  Remember that the cells from the udder were grown "in vitro" for many days. At first the researchers grew the cells in a liquid that included 10% fetal calf serum--a rich diet. Then they switched the cells to a diet with only 0.5% fetal calf serum for five days. That diet was not enough to support cell division--but the cells continued to live in a "resting phase" called G0 (in contrast to G1 and G2) by cell biologists. Such cells can be called quiescent.
  
  
• The researchers report one animal (now known to the world as Dolly), was derived from a combination cell with a nucleus from a mammary cell (a differentiated cell) from an adult sheep.
  
  The authors write that "the fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term." Dolly is the evidence that disproves the idea of irreversible modifications of genetic material in differentiated cells of mammals.
  
  
• Question: What ideas could you test by putting an egg's nucleus in a cytoplasm of a cell taken from an adult?
  
  
• Of the two animals that supplied cells for the combination cell, which supplied the mitochondria?
  
  This was not clear to this writer from the paper. There are three possibilities for the origin of Dolly's mitochondria. 1) The mitochondria came only from the egg cell. 2) The mitochondria came only from the cell derived from udder cells. 3) Dolly's cells have a mixture: some mitochondria originated from the egg cell, and some from the cell derived from the udder cells.
  
  
• If Dolly's nucleus came from one animal and her mitochondria from another, is she really genetically identical to the donor of the nucleus?
  
  Note that while the focus of the popular coverage of the story is the cloning technology, the emphasis of the research report is a disproof of the hypothesis that cell's genes are irreversibly modified by differentiation.

Technique Exquisite

• The researchers report cloning from three different sources of cells, representing three different stages in the development from a fertilized egg to a mature adult. Why would the researchers try a series of cells of increasing age as sources of nuclei?
  
  This is the skiing analogy. At ski hills, there are hills color-coded by difficulty. Green, blue and black are easy, intermediate and difficult. If you start out on the most difficult hill, you're likely to fail before you get a chance to succeed. So most new skiers start easy, and gradually go to the more challenging hills.
  
  The researchers anticipated increasing difficulty of cloning with increasing age of the donor cells--so they tried some young cells to increase the liklihood of success. This also serves as a kind of positive control, to show that their technique and tools could work. In the event that none of the attempts to clone from adult cells succeeded, they could refute the possibility that their technique or tools were flawed.
  
  
• After making the "reconstructed embryos," did the researchers grow them in a test tube or implant them into a surrogate ewe?
  
  
• How was pregnancy monitored? What were the two explanations suggested for the observed decreases in detected pregnancies?
  
  
• How does the rate of fetal death compare between conventional pregnancy and pregnancy from reconstructed embryos?
  
  
• What four kinds of evidence do the authors cite to conclude that the lambs were not born from inadverdent matings?
  
  Note that morphological evidence as well as DNA analysis are used here.
  
  Comments on comparing this paper to the coverage of this paper in the press.
  
  
• How often does the word "clone" appear in the paper by Wilmut and colleagues?
  By definition, a clone is genetically identical to its progenitor. Is Dolly genetically identical to its nuclear donor? Is Dolly a true clone? Do the authors make that claim?
  
  
• How often do the authors talk about "transfer of DNA" in contrast to "transfer of a nucleus"? On February 24 and 25, many of the stories in the press stated that Dolly was cloned by taking "the DNA" from the cell of an adult sheep and transfering it to another cell. The authors use terms such as "the nucleus" and "the genetic material" but do not specifically refer to DNA transfer at all. In fact, of the national media, USA Today got the story right in talking about transfer of the nucleus.
  
  Actually, it was a cell fusion, rather than solely the transfer of a nucleus, but that did not become clear until the paper was published on February 27.
  
  Is the distinction between "DNA transfer" and "nuclear transfer" important?
  
  Sure.The nucleus is an organized organelle, and it has organizational capacity that its DNA does not. This was a transfer of organelles, not just a transfer of DNA. Here, the distinction made ten years ago by Sidney Brenner is important: chemistry-as-information compared to chemistry-as-organization. Transferring an intact nucleus transfers only one copy of all the DNA--but it's a well-organized copy!
  
  Remember to consider this story not just from the "nuclear transfer" point of view but also from the "cytoplasmic transfer" point of view. What is it about the cytoplasm of the egg cell that can activate the expression of genes in the received nucleus?
  
  
• Finally, compare this "cell fusion" to the cell fusion in ordinary fertilization: sperm and egg fuse. The fusion involves two steps: fusing of the cytoplasms (plasmogamy) and later, fusion of the two nuclei (karyogamy). The cell fusion described in this paper would emulate plasmogamy, but not karyogamy, of standard sperm and egg fertilization. The results, if any, of the omission of this step remain to be found.

About Biotech Index