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A new method to clone mice: an important step in the research for applications in the human

December 18, 2000

A simplified method for cloning mice, which has recently resulted in live offspring, has been developed at the Ghent University. Although a few laboratories have been successful in breeding mice by cloning, this is the first report on cloning of an adult mammalian animal in Belgium and using a self-developed purely mechanical method for nuclear transfer. Follows a brief overview of the history and biological significance of cloning and a discussion of some technical aspects and the potential for future applications.���

Cloning: a natural phenomen of reproduction

Cloning is the basic mechanism of reproduction for most of the primitive living organisms and has proven its efficiency throughout the evolution of life. In fact, it is still unknown how and why the sexual mode of reproduction came about, because asexual reproduction has a number of advantages of which the production of copies with proven environmental adaptation is one of the most important. Cloning by transfer of somatic cell nuclei from animals has great potential applications in agriculture, medicine and biological research and this is the main drive for the current research on this topic. The problem with cloning is that the genome of adult somatic cells is already specialised by cell specific programming of DNA. The main challenge and still an important hurdle in the successful application of cloning is the reprogramming of DNA.

History

The first proof that inactive nuclear genes were not lost or permanently inactivated in differentiated cells came from cloning experiments in frogs already forty years ago. Twenty years later, live births in mammals obtained by transfer of nuclei from embryonic stem cells were reported. The breakthrough, however, came with the birth of Dolly, the first sheep to be cloned from adult somatic cells. This provided the final proof that adult somatic cells are totipotent and can be reprogrammed. The announcement of the birth of Dolly provoked a sensational shock leading to all kind of speculations on the possibility of cloning human beings in the near future. This public turmoil obscured the great potential this new technology has for basic research in the fields of ageing, cancer, X-chromosome inactivation and imprinting, the promise it holds for commercial application by gene targeting in livestock and last but not least, the tantalising prospect it brings for the production of customized stem cells for therapeutic uses.���

Essential steps in cloning

Different types of somatic cells can be used for nuclear transfer, provided that they are in a resting state. The nucleus of these cells are transferred to cytoplasm of a mature oocyte, i.e. an oocyte which is ready to be fertilized and has geared up the biochemical pathways for reprogramming and cleaving.
Before bringing the somatic cell into contact with the oocyte, the nucleus of the oocyte has to be removed.
Several methods are currently used for nuclear transfer in farm and laboratory animals. They can be divided into two groups, fusion and injection, depending on how the donor nucleus is brought into the recipient cytoplasm. In one procedure, the donor cell (karyoplast) is brought into contact with the membrane of the oocyte (the recipient cell or cytoplast) and fusion of the two membranes is effected by an electric pulse (electrofusion) after which the nucleus enters the cytoplasm. In the injection method the cellular membrane surrounding the donor nucleus has to be destroyed before or during mechanical injection of the nucleus into recipient cytoplasm.
Following the insertion of the donor nucleus, the recipient cell has to be activated to start the cleaving process. For reproductive cloning, the resulting embryo has to be transferred into the uterus of a surrogate mother.

Cloning of mice

We have developed a simplified method for cloning mice by using a conventional mechanical method of nuclear transfer. The few laboratories which were successful in cloning mice have done so by using a piezo-driven mechanical injection method. This technique is based on piezo-effect generated vibrations of the micro-pipettes which are used for oocyte enucleation and for injection of the donor nucleus. These vibrations allow penetration of the zona pellucida with a blunt pipette while the oocyte membrane can be easily penetrated with minimal risk of damage to the injected oocyte. However, in addition to the need for expensive piezo-effect generators this technique may require a number of technical tricks and additional equipment.
Conventional techniques of injection have been successfully established and perfected for intracytoplasmic injection of spermatozoa into human and mouse oocytes. This technique is less demanding with regard to the type of equipment for micromanipulation and if applied in combination with partial zona dissection (PZD) allows a broader range of shape and geometry of the injection needles. On the other hand, with this technique it is frequently needed to aspirate a rather big volume of oocyte cytoplasm into the injection pipette to brake the oolemma, which could inflict some damage to the oocyte structures and reduce the oocyte survival rate.
Considering the economic advantages of the conventional technique and the availability of equipment and extensive experience in our lab we adapted the technique for mouse cloning. After several months of research and several trials we recently succeeded in producing live offspring.

Importance of cloning

There are three main goals of cloning: reproduction of animals, production of embryonic stem cells and biological research.
Reproductive cloning holds a great promise for agriculture and also for bioengineering by gene targeting of animals. The use of transgenic livestock for the production of therapeutic proteins in milk is now well established.���Preservation of rare or even extinct species is another future application of reproductive cloning. A major problem that remains to be solved is the rather low efficiency of the technique and the until now unexplained high perinatal mortality of cloned animals.
The purpose of our experiments was to develop a feasible and reliable method to clone somatic cells as a stepstone for the study of a number of biologic questions and the production of embryonic stem cells.���

Biological research

Cloning can be helpful in investigating a number of fundamental biological questions that remain to be answered such as:
* Which factors are involved in genetic reprogramming of DNA
* Can all somatic cells be reprogrammed
* How and when does inactivation of the X chromosome occur
* Do cloned somatic cells keep their genetic imprinting (maternal and paternal zygotic genomes differ due to epigenetic imprinting resulting in their differential expression during embryogenesis).
* How old are cloned organisms. Ageing is a complex phenomenon where genetic and structural damage of DNA play a role that remains to be determined
* What makes a cell to differentiate

Practical applications

One of the main spin-offs of the cloning technology is the production of stem cells.

Stem cells
Stem cells could be derived from a number of sources
* From early embryos (blastocysts) created by in vitro fertilisation, either ‘spare embryos’ which are not needed for infertility treatment or embryos created specifically for research
* From early embryos created by cloning
* From the germ cells or organs of an aborted fetus
* From the blood cells of the umbilical cord at the time of birth
* From some adult tissue (bone marrow)
* From mature adult tissue reprogrammed to behave like stem cells

Cells from young embryos have the potential to differentiate into all kind of tissues whereas cells from adult tissues will be limited in this respect. It may become possible in the future to reprogramme adult cells to behave like stem cells but at the moment this remains largely hypothetical. Besides many technical hurdles to be overcome before the potential benefits of stem cell techniques could be realised, there are also some ethical constraints with regard to the creation of embryos for therapeutic use.���The use of any embryo for research purposes is thought to be unethical by part of the public opinion on the grounds that an embryo should be accorded full human status from the moment of its creation. Others argue that the respect due to the embryo is relative and is mainly determined by its stage of development and its final destination and that the respect should be weighed against the potential benefit arising from the proposed research. An unanswered question is whether an embryo obtained by nuclear transfer deserves the same respect as an embryo obtained by fusion of male and female gametes. The benefits of being able to develop an individual’s own cells to create a new source of cells for their own future treatment might, however, be a strong ethical argument. Anyway, research into cell nuclear replacement might offer a means of producing compatible tissue for treatments and it may offer the only means of learning about the mechanisms for reprogramming of adult cells. In the long term stem cells might be used in the treatment of a wide range of disorders such as diabetes or Parkinson’s disease by replacing cells that have been damaged.

Gametes
Absence of sperm or eggs is an important reason for infertility, that currently can only be helped by sperm or oocyte donation. Reproductive cloning is not a solution for the couple and the prospect of cloning human beings is, for the time being, deemed not to be ethically acceptable. The eventual purpose of our research is to investigate whether gametes can be constructed by nuclear transfer technology, which could restore biparental reproduction. It is indeed biologically possible to induce meiosis in somatic cells after nuclear transfer into immature oocytes. Before this can be put into practice, however, a number of technical aspects will have to be addressed in animal experiments. It would be wonderful to restore the possibility of sexual reproduction by technology derived from asexual reproduction. The construction of fertile gametes from somatic cells, e.g. skin cells would be a landmark in the history of reproductive medicine and a big step in the treatment of infertility.

Ghent University