Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse

Abstract

Addressing the fundamental questions of nuclear equivalence in somatic cells has fascinated scientists for decades and has resulted in the development of somatic cell nuclear transfer (SCNT) or animal cloning. SCNT involves the transfer of the nucleus of a somatic cell into the cytoplasm of an egg whose own chromosomes have been removed. In the mouse, SCNT has not only been successfully used to address the issue of nuclear equivalence, but has been used as a model system to test the hypothesis that embryonic stem cells (ESCs) derived from NT blastocysts have the potential to correct—through genetic manipulations—degenerative diseases. This paper aims to provide a comprehensive description of SCNT in the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very challenging and inefficient procedure because it is technically complex, it bypasses the normal events of gamete interactions and egg activation, and it depends on adequate reprogramming of the somatic cell nucleus in vivo. Improvements in any or all those aspects may enhance the efficiency and applicability of SCNT. ESC derivation from SCNT blastocysts, on the other hand, requires the survival of only a few successfully reprogrammed cells, which have the capacity to proliferate indefinitely in vitro, maintain correct genetic and epigenetic status, and differentiate into any cell type in the body—characteristics that are essential for transplantation therapy or any other in vivo application.

Introduction

Somatic Cell Nuclear transfer (SCNT) was developed over 55 years ago by Briggs and King [1] in amphibians to address the question of nuclear equivalency i.e., whether the nucleus of a somatic cell is genetically equivalent to the nucleus of a zygote. Since then, SCNT has been instrumental and indeed a fruitful approach in showing that differentiation is a reversible process and that an adult nucleus has the potential to turn back into an embryonic state of development that has been retained during lineage commitment. The generation of Dolly, the sheep, via SCNT [2], was the first experiment to demonstrate that adult somatic cell nuclei retain the potential to direct the development of an animal when placed in the environment of an oocyte. The resulting ‘clones’ are genetically identical to the animals that donate the somatic cells in the SCNT procedure. A year after Dolly’s birth, successful cloning of the mouse from adult cells was reported [3] and subsequent reports showed that terminally differentiated cell types such as lymphocytes and post-mitotic neurons [4], [5], [6] still retain the potential to direct embryonic development and give rise to an entire organism. In addition to sheep and mouse, more than 15 other species, including cattle, goat, pig, cat, and dog have been successfully cloned [7]. More recently the derivation of embryonic stem cells (ESCs) from cloned rhesus macaque blastocysts was also reported [8].

Ten years after the generation of the first cloned mouse, Cumulina, from cumulus cells [3], mice have been cloned using a number of cell types including embryonic stem cells [9], [10], [11], fibroblasts [12], [13], [14], Sertoli cells [15], primordial germ cells [16], [17], natural killer T cells [18], hematopoietic stem cells [19], neurons and neuronal stem cells [20]. Several conclusions have been made from these experiments. Firstly, almost all cell types under appropriate conditions should be able to be used as donors for SCNT. Secondly, the efficiency of the SCNT technique varies considerably for different cell types. For example, Sertoli cells are more efficiently cloned than cumulus cells [3], [15]; adult hematopoietic stem cells are less efficiently cloned than cumulus, Sertoli or fibroblast cells [19], [21]; embryonic stem cells (ESCs) are more efficiently cloned than any other cell type [22]. The observed variability in the cloning efficiency has been attributed to the differentiation state of the donor cell, the cell cycle status of the donor cell, and technical aspects of the procedure. Thirdly, even in the best case scenario, the efficiency of SCNT is extremely low. In some cases, e.g., the cloning of terminally differentiated cells of the lymphoid lineage, the process is so inefficient that cloned animals have not been generated following the classical NT procedure. To overcome this problem, a modified two-step SCNT procedure has been developed. This procedure involves the derivation of embryonic stem cells from NT blastocysts—NT-ESCs—that are injected into tetraploid blastocysts, which in turn give rise to animals derived entirely from the injected NT-ESCs. This two-step procedure enabled the generation of viable clones from terminally differentiated murine B and T lymphocytes [4] and post-mitotic olfactory neurons [5], [6].

Mouse SCNT, despite its successful application by various labs, is a very challenging technique, much more challenging than SCNT in other species, such as the bovine. One technical aspect that makes mouse SCNT more challenging relates to the low success of donor nucleus transplantation by fusion, which is most commonly used with domesticated species. The use of fusion in mouse cloning results in premature egg activation, which impairs the ability of the cloned egg to develop properly. The adaptation of the piezo impact drive unit to transfer the somatic cell nucleus into the egg’s cytoplasm [3] was pivotal in making SCNT more accessible to several laboratories in which mouse SCNT is now a standard practice. Although the use of piezo-assisted injection presents its own caveats—it introduces extra-stress and damage to the egg and somatic cell nucleus—it has been invaluable in bypassing the issues of premature egg activation.

In addition to issues related to the introduction of the donor nucleus into the enucleated egg, SCNT is faced with numerous other challenges that have a major impact on its efficiency. One such challenge is the strain of mouse used, both in terms of the source of donor nuclei and source of eggs. Very few strains have been used successfully, mainly those of hybrid F1 and 129 backgrounds [23], [24], although recently the cloning of an outbred strain was reported [25].

Finally, SCNT is hampered not only by its low efficiency but also by the abnormal phenotype of the clones [26]. Certain phenotypes of cloned mice have been associated with the donor cell type e.g., cloned mice derived from Sertoli cells die prematurely and exhibit hepatic failure and tumors, whereas those derived from cumulus cells suffer from obesity [27], [28]; other phenotypes, such as large offspring syndrome, are common to all clones. In addition, all clones—those that die prematurely, as well as those surviving to adulthood—have been demonstrated to exhibit faulty gene expression and DNA methylation patterns [29], [30], [31]. However, these abnormalities are not passed onto the offspring, suggesting that these abnormalities are of epigenetic rather than genetic nature [27].

In contrast to the inefficiency by which animal clones are generated, NT-ESCs are derived much more efficiently [22]. In addition, in contrast to the abnormal phenotype of all clones, NT-ESCs have been shown to be virtually the same as ESCs derived from fertilized blastocysts, as assessed by global expression profiles and ability to generate chimeric mice and mice following injection into tetraploid blastocysts [32], [33]. Supported by the evidence of equivalency of NT-ESCs to ESCs from fertilized blastocysts, several applications have been suggested for NT-ESCs, including: (i) The correction of genetic and degenerative disorders, such as Parkinson’s, Alzheimer’s, diabetes and others. The proof of principle experiment has already been successfully performed in mice [34]. (ii) The preservation of genes from infertile mice generated by methods such as large-scale ethyl-nitrosourea (ENU) mutagenesis. (iii) The generation of unlimited source of nuclei from valuable mouse strains—the propagation of those strains can then be performed without the use of frozen gametes or embryos from that strain.

Despite the numerous challenges associated with the technique, mouse SCNT has been invaluable in approaching complex biological questions such as reprogramming, imprinting, early embryonic development, and cancer. Even though new methods such as in vitro reprogramming of somatic cells using a combination of transcription factors have been developed [35], [36], [37], for all the above reasons, SCNT will continue to be a useful technique. In this paper, we provide protocols for mouse cloning that have been used in our laboratory over the past 7 years and which have provided the basis for many of the studies cited above. Additionally, we describe methods used to derive ESCs from NT blastocysts.