Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection

Abstract

Embryonic stem (ES) cells provide a unique tool for producing specifically designed mutations in mice. Here, we describe an alternative approach toward the generation of mice which are derived completely from ES cells (ES mice), as judged by glucose phosphate isomerase (GPI) analysis, without prior passage through the germline. By injecting wild-type and mutant ES cells into tetraploid blastocysts, viable and fertile ES mice were generated, suggesting that totipotency of ES cells was not affected by long-term culture and experimental manipulation in vitro. When ES cell clones harboring a lacZ reporter gene introduced by either targeted insertion or a gene-trap approach were used, the expression pattern of the lacZ gene in ES fetuses was identical to that of fetuses that were derived from breeding of chimeric mice. Thus, this technique can be considered as a useful and rapid approach to produce fetuses and mice directly from ES cells carrying predetermined genetic changes and offers many applications for studies in molecular genetics and developmental biology.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

“…Using specific culture conditions, ES cells can be maintained in an undifferentiated state throughout multiple cell divisions, even after many passages and following genetic ma- nipulations (3)(4)(5)(6). An important feature of ES cells is their ability to be induced to enter a program of differentiation in vitro.…”

“…When transferred to a suspension culture, they spontaneously form aggregates of differentiating cells known as embryoid bodies (EBs) (3,(7)(8)(9)(10)(11)(12). Within the EBs, a variety of early embryonic lineages (hematopoietic, neuronal, vascular endothelial, cardiac and skeletal muscle) can be identified by morphological, immunohistochemical and RT-PCR analysis (3,6,(12)(13)(14)(15)(16). Studies of developmental gene expression in EBs indicate that derivatives of the three germ layers formed during gastrulation and early organogenesis are present in EBs.…”

« Clonage thérapeutique » : de la théorie à la pratique

“…La mort précoce par anémie des souris dont les gènes codant pour l'Epo ou pour son récepteur avaient été invalidés n'avait pas permis, dans les expériences initiales, d'identifier d'autres actions importantes de l'Epo. Un défaut de développement cardiaque avait été rapporté chez ces souris en 1999 [4] et, récemment, un article publié par le groupe de T. Noguchi montre que l'EpoR est aussi impliqué dans la formation du cortex cérébral [5]. Ces auteurs montrent d'abord par immunohistochimie une forte expression de l'EpoR, à partir de 10,5 jours de gestation, dans toutes les couches neuro-épithéliales (zones germinatives) du cerveau, dont le cortex.…”

“…Avant de « réparer ces cellules ES » encore fallait-il s'assurer de leur « pluripotence », c'est-à-dire de leur capacité à contribuer in vivo à la constitution de tous les tissus embryonnaires. Or, les cellules ES ayant perdu la capacité de donner les annexes (placenta) requises pour le développement d'un embryon viable, elles doivent être « complémen-tées », ce qui est fait par leur agrégation à des cellules embryonnaires tétraploïdes (obtenues par électrofusion de cellules embryonnaires au stade deux cellules) [5]. Dans ces conditions, tous les tissus des embryons dérivent des cellules ES et les annexes des cellules tétraploïdes.…”

Trophoblastic vesicles as carriers of embryonic cells for mammalian cloning

“…Mutant mice strains can be generated by producing animals derived from transgenic embryonic stem cells, without the intermediate step of chimaerae formation and their further breeding (Wang et al, 1997). Also, animals originating from a given cell line constitute a clone, so that this method can be an alternative way to clone mammals, possibly including livestock species.…”