Oocyte nuclei

An example of an RNA injection/immunoprecipitation experiment is shown in Fig. 2. In this experiment, a mixture of radiolabeled snRNAs were coinjected into oocyte nuclei. Oocyte fractionation over time followed by electrophoresis of the RNAs in both the nuclear and cytoplasmic compartments (Fig. 2A) revealed the nucleocytoplasmic distributions of the injected RNAs. The RNAs present in the nuclear fractions were extracted and immunoprecipitated (Fig. 2B and C) using antibodies recognizing either the m G cap (of the precursor snRNAs) or a hypermethylated m G cap (of the mature snRNAs). This and other experiments (Terns and Dahlberg, 1994 Terns et ai, 1995) demonstrated that U3 RNA and other small nucleolar RNAs differ from most RNA polymerase Il-transcribed RNAs in that they are not exported from the nucleus to the cytoplasm. Furthermore, unlike spliceosomal RNAs, which undergo hypermethy-lation of their m G cap structures within the cytoplasm (Mattaj, 1986), small nucleolar RNAs receive their trimethylated cap structures within the cell nucleus. 

Many old and new investigations have shown that the structure of chromosomes, in their different states (during mitosis and interphase), is based on fibrillary formations whose diameter varies from 100 to 600 A. These investigations have been conducted on widely different objects cell nuclei of Tradescantia and of bees, liver cell nuclei, oocytes and erythrocytes of Triturus, and so on. 

Fig. 2. Methods for production of identical catde. (a) Embryo spHtting. A microdissection knife is used to cut an early embryo into two identical halves, (b) Embryo cloning. A cell from a morula stage embryo is removed and then inserted into an oocyte that has had its nucleus removed. EoUowing fusion of the two cells the newly produced one-ceU embryo is identical to the original morula. The procedure can be repeated to give multiple copies of the original...

The first cell cycle of the mouse embryo differs in many respects from the second and the following cell cycles. It is characterized by a long Gl phase that starts after the penetration of the spermatozoon or artificial activation of the oocyte. During this period the chromatin of the oocyte completes the second meiotic division and forms the female pronucleus. At the same time, in the fertilized egg, the highly condensed chromatin of the sperm nucleus decondenses and sperm-specific proteins, protamines, are replaced by histones. After the initial sperm chromatin... 

Hunt The most extreme case is an oocyte, which has a giant nucleus and very little DNA. This must just be because there are an awful lot of proteins that belong in the nucleus, and it fills up. 

Direct injection of cDNA into the oocyte nucleus can lead to high levels of expression (note that this technique may require centrifugation of the oocytes to orient the nuclei as injection targets see... 

You have extracted the nucleus from an oocyte. You have a nucleus and the rest of the cell. Neither of these two components is alive. Now you insert again the nucleus into the cell - or, in this respect - into a different enucleated cell (as in the cloning experiments). You get a living cell. Can this be taken as a demonstration for the emergence of life, to namely that hfe can be generated out of non-living components ... 

SCNT is a cloning strategy, originally reported by Campbell, et al. [33], in which nuclei are isolated from a donor s somatic cells, such as fibroblasts, and are transferred into enucleated oocytes from female donors [33]. By mechanisms yet to be uncovered, the cytoplasm of the oocytes reprograms the chromosomes of the somatic cell nucleus and the cloned cells develop into blastocysts, from which ESCs can be derived [34]. One of the landmarks of SCNT is the potential to generate isogeneic ESCs, carrying a set of chromosomes identical to that of an individual, and therefore unlikely to be rejected after transplantation into that individual [35]. 

Lampbrush chromosome. Giant diplo-tene chromosome found in the oocyte nucleus. The loops that are observed are the sites of extensive gene expression. 

Different classes of RNAs, including mRNAs, tRNAs, and U snRNAs, are transcribed and processed within the nucleus and then exported to the cytoplasm by multiple pathways. Competition experiments with microinjected Xenopus oocytes have revealed that tRNA, mRNA, U snRNA and ribosomal subunits all use saturable pathways, but none of them saturated the export of others (Jarmolowski et al, 1994). For instance,... 

Starting in 1993, reports have appeared that in rat striatum and nucleus accum-bens D2-like autoreceptors, when activated, enhanced removal of dopamine from the extracellular space by means of the dopamine transporter (DAT) as studied by voltammetry (Table2). D2 receptor activation also increased [3H]-dopamine uptake in Xenopus oocytes transfected with both the human D2 receptor and the human DAT (Mayfield and Zahniser 2001), as well as the uptake of a fluorescent DAT substrate in human embryonic kidney cells equally transfected with both the human D2 receptor and the human DAT (Bolan et al. 2007). In accord with this autoreceptor-transporter connection, clearence of dopamine from the striatal extracellular space in vivo was reduced in D2 receptor knockout mice (Dickinson et al. 1999). Unfortunately, however, the data in the literature are by no means consistent. No effect of D2 receptor activation on the DAT was observed in another rat striatum study (Prasad and Amara 2001), in synaptosomes from rat and human neocortex (Feuer-stein, unpublished observations), and in PC 12 cells that possess D2 receptors and the DAT (Pothos et al. 1998). In a second study with D2 receptor knockout mice, uptake of dopamine in the striatum was increased rather than decreased (Schmitz et al. 2002). The reason for these discrepancies is not clear. It has been suggested that the clearance measurements by means of voltammetry were not appropriate for assessment of the function of the DAT (Prasad and Amara 2001). 

The rate of elimination of a drug from the body. Process that involves removing the nucleus from an adult cell, transferring it to an unfertilized oocyte, destroying the genome of the oocyte and allowing the resulting cloned cell to develop. 

MEIOSIS The process by which a eukaryotic cell nucleus (in which the DNA has been replicated only once) undergoes two coordinated divisions that yield four cells, each having ploidy half that of the original cell in higher animals, meiosis provides for the production of haploid sperms or eggs from diploid spermatocytes or oocytes. (See also MITOSIS)... 

Transfection efficiency is dependent on mitotic activity, as cells prevented from going into mitosis after transfection express transgenes much less efficiently than proliferating cells. In search for an explanation, the transport of plasmids across the nuclear membranes has been studied. Plasmids injected into the cytoplasm of quiescent human fibroblasts are not expressed, in contrast to plasmid injected into the nucleus. This has been found to be true for the cationic lipid-based systems as plasmid injected into the cytoplasm of Xenopus oocytes is not expressed, unlike that injected into the nucleus, it must be concluded that the plasmid must dissociate from the cationic lipids before entering into the nucleus. 

Human embryonic stem cells (hESCs) were first derived from the inner cell mass (ICM) from the blastocyst stage (-100-200 cells) of embryos generated by in vitro fertilization [35,36]. In humans the blastocyst is an early-stage embryo, approximately 4 to 5 days old. The blastocyst can be formed by means of either in vitro fertilization or somatic cell nuclear transfer, in which the nucleus of a somatic cell is combined with an enucleated oocyte. Methods have been developed to derive hESCs from the late morula stage (30-40 cells) and from arrested embryos (16-24 cells incapable of further development), and more recently from single blastomeres isolated from 8-cell embryos (37). 

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