Plasticity of the inner cell mass in mouse blastocyst is restricted by the activity of FGF/MAPK pathway.

In order to ensure successful development, cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or inner cell mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential cell fate decisions. We revealed that TE-deprived ICMs derived from 32-cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.

RNA interference by production of short hairpin dsRNA in ES cells, their differentiated derivatives, and in somatic cell lines.

dsRNA of several hundred nucleotides in length is effective at interfering with gene expression in mouse oocytes, pre-implantation embryos, and embryonic stem (ES) cells but is not as efficient in differentiated cell lines. Here we describe a method to achieve RNA interference in totipotent and differentiated ES cells together with a wide range of other mammalian cell types that is both simple and efficient. It utilizes a linearized plasmid that directs the expression of a hairpin RNA with a 22-nucleotide-paired region. This molecule has a 13-nucleotide 5' overhang that would be subject to capping on its 5' phosphoryl group and thus differs from the ideal structure suggested for effective small interfering RNAs. Thus, it appears either that the structure of small inhibitory RNA molecules may not need to be as precise as previously thought or that such a transcript is efficiently processed to a form that is effective in interfering with gene expression.

The possible role of cell cycle stage in mammalian cloning.

Progress in mammalian cloning started from cloning embryos (of mice, rats, rabbits, sheep, goats, pigs, cattle and rhesus monkeys) and culminated in obtaining clones of sheep, cattle, pigs and mice from adult somatic cells. Knowing the relationship between the cell cycles of the recipient and the donor of cell nucleus in embryonic cloning by nuclear transfer one can adjust the phases of the cell cycle properly. Metaphase II recipients accept G1 (in most species) or G2 donors (in the mouse). Interphase recipients can harbour nuclei in all stages of cell cycle. Relatively little is known about somatic cloning. Two attitudes are applied: either the donor is in the G0 phase or the recipient is in a prolonged MII phase.

Transcription and DNA replication of sperm nuclei introduced into blastomeres of 2-cell mouse embryos.

The aim of this study was to investigate the behaviour of sperm nuclei in the cytoplasm of the 2-cell mouse embryo. To this end, we produced hybrids between anucleate fertilised oocyte fragments and blastomeres of the 2-cell embryos. When sperm nuclei at the stage of decondensation or recondensation were introduced into blastomeres the development of male pronuclei was usually retarded and they never reached the size of the blastomere nuclei. These abortive male pronuclei were unable to initiate transcription but they were capable of synthesising DNA. The majority of sperm nuclei introduced into blastomeres as early male pronuclei developed normally and reached the size of the blastomere nuclei. They synthesised DNA simultaneously with blastomere nuclei and were transcriptionally active. In addition they participated in the cleavage division of hybrid cells. This shows that the very early male pronucleus when transmitted from the oocyte cytoplasm to the blastomere cytoplasm can respond positively to the new cytoplasmic factors, i.e. it undertakes both DNA replication and transcription according to the time schedule characteristic of the second cell cycle.