Defining Critical Parameters of Mouse Cloning

自1986年以来，哺乳动物克隆已经成为可能，但其背后的生物学原理仍不清楚。与这种缺乏基本认识相关的是，无论物种(牛、山羊、猪、绵羊和老鼠)，活体出生的克隆后代的获取频率一直很低：充其量约为2%。到目前为止，大多数努力都集中在克隆家畜动物上，但这些大型动物并不是方便的实验模型。小鼠特别适合于这一目的，因为它易于操作、维护和繁殖，妊娠期短，而且小鼠的遗传学处于先进状态。但直到最近，还没有可重复使用的小鼠克隆技术。我们研究的总体目标是了解与小鼠核移植后重编程相关的生物学现象。在初步研究中，已经开发出一种可重复性的方法，通过核移植来克隆可存活和可生育的小鼠。它已被应用于卵丘细胞、尾部衍生细胞、胚胎干细胞；雌性和雄性小鼠；以及近交系129/O1a。有证据表明，处于细胞周期不同阶段的细胞可以用作核供体。具有胚胎干细胞特性的细胞系是从核移植产生的囊胚中衍生出来的。总体而言，我们已经用这种方法培育了250多只活的克隆小鼠。这项技术最近在洛克菲勒大学重新建立起来。为了验证成熟小鼠的核可以被卵母细胞重编程并支持克隆小鼠的完全发育的假说，并由此确定小鼠克隆过程中的关键参数，提出了以下具体目标：1)成熟淋巴细胞和有丝分裂后神经元的核是否可以重编程？2)供体细胞周期的阶段是否影响核的重编程能力？3)核移植后的重编程对端粒长度有何影响？4)通过核移植产生的囊胚能否建立多能ES细胞系？总而言之，这些实验应该可以解决哺乳动物克隆生物学中的关键和突出问题。他们还应该验证鼠标作为模型系统的选择。这项拟议的研究有望提高这项技术的效率，尽管这不是主要目标。

Mammalian cloning is possible since 1986, yet the underlying biology remains obscure. Related to this lack of fundamental understanding is the consistently low frequency with which live-born cloned offspring are obtained, irrespective of the species (cattle, goat, pig, sheep and mouse): it is at best approximately 2 percent. To date the majority of efforts have focused on cloning livestock animals, but these large animals are not convenient experimental models. The mouse is particularly suitable for this purpose, because of the ease of handling, maintenance and breeding, the short gestation period, and the advanced state of mouse genetics. But until recently reproducible techniques for mouse cloning did not exist. The overall aim of our studies is to understand the biological phenomena associated with reprogramming following nuclear transfer in mice. In preliminary studies, a reproducible method has been developed to clone viable and fertile mice by nuclear transfer. It has been applied to cumulus cells, tail-derived cells, embryonic stem cells; to female as well as male mice; and to an inbred strain, 129/Ola. Evidence has been provided that cells at various stages of the cell cycle can be used as nucleus donors. Cell lines with embryonic stem-cell properties have been derived from blastocysts generated by nuclear transfer. In total, we have generated greater than 250 live-born cloned mice with this method. The technology has been re-established at The Rockefeller University following a recent move. To test the hypothesis that the nucleus of a differentiated cell in the adult mouse can be reprogrammed by an oocyte and support full development to term of a cloned mouse, and to define thereby the critical parameters that underly the cloning process in mice, the following Specific Aims are proposed: 1) Can nuclei from mature lymphocytes and postmitotic neurons be reprogrammed? 2) Does the stage of the donor cell cycle influence the ability of nuclei to be reprogrammed? 3) How is telomere length affected by reprogramming after nuclear transfer? 4) Can pluripotent ES cell lines be established from blastocysts generated by nuclear transfer? Taken together, these experiments should resolve critical and outstanding issues in the biology of mammalian cloning. They should also validate the choice of the mouse as a model system. The proposed research is expected to improve the efficiency of this technique, although this is not the primary objective.