Teruhiko Wakayama

Improvement of Mouse Cloning from Any Type of Cell by Nuclear Injection

Somatic cell nuclear transfer (SCNT) technology has become a useful tool for animal cloning, gene manipulation, and genomic reprograming research. The original SCNT was performed using cell fusion between the donor cell and oocyte. This method remains very popular, but we have recently developed an alternative method that relies on nuclear injection rather than cell fusion. The advantages of nuclear injection include a shortened experimental procedure and reduced contamination of donor cytoplasm in the oocyte. In particular, only this method allows us to perform SCNT using dead cells or naked nuclei such as those from cadavers or body wastes. This chapter describes a basic protocol for the production of cloned mice by the nuclear injection method using a piezo-actuated micromanipulator as well as our recent advances in SCNT using noninvasively collected donor cells such as urine-derived somatic cells. This technique will greatly help not only SCNT but also other forms of micromanipulation, including sperm microinjection into oocytes and embryonic stem cell injection into blastocysts.

Nuclear Transfer Embryonic Stem Cells as a New Tool for Basic Biology

Recently, induced pluripotent stem (iPS) cells have become a very hot topic, and the popularity of nuclear transfer embryonic stem (ntES) cells has declined due to ethical problems in addition to the extremely difficult techniques required. However, ntES cells have a unique potential: some research designs can be approached only with this procedure and cannot be replaced by any other procedure, including iPS cell technology.We previously showed that ntES cells can be established with a tenfold higher success rate than the production rate of cloned mice, including a variety of mouse genotypes and cell types, even though it may be more difficult to generate the clones directly. Moreover, ntES cells were identical to the ES cells derived by fertilization in terms of their global gene expression and their differentiation potential. In addition, several reports have demonstrated that ntES cells can be used in regenerative medicine, such as in the treatment of Parkinson disease or of immunodeficient mice. In addition, by combining the cloning and the ntES cell techniques, this approach could be applied to fertility treatments using somatic cells instead of gametes. Furthermore, live mice now can be generated from frozen dead bodies, suggesting that extinct animals, such as the mammoth, can be possibly resurrected by this technology if nondamaged nuclei are retrieved from the permafrost. Thus, although there remain many cloning issues in ntES technology, it has the potential to become a powerful new research tool with broad-based applications in the study of basic biology.