discussions during the course of this study, Michael Malim for the GPV-RRE construct, Bettina Abel and Andrea Bunk for technical assistance, and ARIAD Pharmaceuticals Inc. for the regulation kits used. Heterokaryon Assay To analyze the nucleocytoplasmic shuttling of Rev, 66104 HeLa cells, transiently transfected with various Rev expression plasmids, were cocultured at 24 h post-transfection with 86104 murine NIH3T3 cells on coverslips. The next day, protein expression was blocked by using 100 mg/ml cycloheximide and 20 nM leptomycin B to inhibit CRM-1 specific nuclear export. Cell fusion was performed by using PEG1500 according to the manufacturer’s protocol and cells were cocultured in cycloheximide- and LMB-containing Somatic cell nuclear transfer is one of the 
best methods available to reprogram differentiated cells so as to render them totipotent. The somatic nucleus is transferred into an enucleated oocyte that subsequently drives a deterministic reprogramming process, as opposed to the stochastic processes induced by reprogramming using either forced expression of defined factors, 1 Uncoupled Differentiations after SCNT cell fusion, or nuclear incubation with cell extracts to induce pluripotency in vitro. In all cases, cell fate reprogramming relies on the epigenetic remodelling of the initial chromatin architecture, either through DNA methylation, histone modifications, or nucleosome spacing, which leads to gene repression/derepression and thus to molecular changes. While all nuclear reprogramming techniques suffer from inefficiency, SCNT is the most efficient so far. It is also the only method that addresses re-differentiation processes in vivo all the way through development to term. We examined re-differentiation processes prior to implantation using bovines because SCNT effectiveness is PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22190027 relatively high in this species and their pre-implantation development period is long. Indeed, although 20% to 60% of SCNT attempts in bovines result in blastocysts, the technique only results in live birth 1% to 10% of the time due to lethal defects and faulty reprogramming. Although SCNT blastocysts could perform equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and TKI 258 web gastrulation defects were reported prior to implantation. However, past studies have not examined more than one of these processes, elongation, gastrulation, or implantation, at a time. Indeed, no previous work has addressed elongation and gastrulation concomitantly and, even though SCNT conceptuses without embryonic discs have been found to elongate, as do trophoblastic vesicles, none have been subjected to molecular analyses. We studied SCNT conceptus development prior to implantation to understand the embryonic/extra-embryonic interactions at work in differentiating blastocysts following their transfer to temporary recipient cows. In particular, we examined the elongation and gastrulation of Day 18 conceptuses. We based our descriptions of developmental patterns on previous observations of tissue differentiation and interactions from Day 12 to Day 25 that we gleaned from studies of artificial insemination or in vitro embryo production . Based on these past results, we expected D18 extra-embryonic tissues should display a filamentous shape while D18 gastrulating tissues should harbour a primitive streak. In addition, extra-embryonic and embryonic differentiations always appear to be synchronised and interdependent: i) the EE tissu
