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Recently, we demonstrated that if NT of the somatic cells is performed prior to the activation and the antimitotic treatment, blastocyst rates can be increased to values similar to those obtained with ME cytoplasts Costa-Borges et al. However, the development to term of the resultant embryos was not tested in this study. In addition to the use of alternative methods of oocyte enucleation, enhancing the potential of the resulting cytoplasts for nuclear reprogramming by treatment with epigenetic modifier drugs may also help to improve the success of the SCNT technique.

In this sense, several studies have revealed that the transient treatment of mouse SCNT embryos with a histone deacetylase inhibitor HDACi like trichostatin A TSA or valproic acid VPA can significantly enhance the potential of the clones to develop in vitro and to term Kishigami et al. In our study, we investigate the effect of the combination of different enucleation approaches with the use of an HDACi on the success of the SCNT technique.

To this aim, we compared the in vitro embryonic development, acetylation status of histone H3 lysine 14 H3K14 , blastocyst quality, ntESCs establishment potential, and embryonic development to term, between VPA-treated mouse cloned embryos generated by ME, AE, or IE procedures. Acetylation level of H3K14 in VPA-treated cloned mouse embryos produced by three different enucleation procedures at different time points after nucleus injection. On the right: SCNT embryos produced by injection of a cumulus cell nucleus into non-manipulated oocytes, which were then subjected to the induced enucleation IE protocol combined with the mechanical aspiration of the PB2.

The majority of oocytes in the ME group survived the removal of spindle chromosomes complex In the AE protocol, In the IE group, Table 2 Comparative in vitro development of cloned embryos produced from cytoplasts prepared by mechanical ME , assisted AE , or induced IE enucleation procedures. The two-cell and morula rates of cloned embryos generated from cytoplasts prepared by ME and AE procedures were very similar and significantly higher than those obtained in IE group. The highest rates of blastocyst formation were achieved with cytoplasts prepared by AE, although no differences were found when compared with ME.

Blastocyst rates were also equivalent between the IE and the ME groups. The total and inner cell mass ICM mean cell numbers in control parthenogenetically activated and in cloned blastocysts produced by the three enucleation procedures are shown in Table 3. Although the total cell number was significantly higher in parthenogenetic blastocysts when compared with the three groups of cloned embryos, no differences were observed in the mean number of ICM cells among any of the groups. Table 3 Mean total and inner cell mass cell numbers in control parthenogenetically-activated oocytes PA and in cloned blastocysts produced from enucleated oocytes prepared by mechanical ME , assisted AE , and induced IE enucleation procedures.

Introduction

ESC lines positive for the three pluripotency markers analyzed could be derived from all groups of control and cloned embryos Table 4. Table 4 Establishment of embryonic stem cell ESC lines from control in vivo fertilized and parthenogenetically activated PA blastocysts and from cloned blastocysts produced from cytoplasts prepared by mechanical ME , assisted AE , or induced IE enucleation procedures. A SCNT blastocyst attached to the feeder cell monolayer.

B A 7-day outgrowth. C A day ntESC-like colony. D ntESC-like line. E Hoechst counterstaining.

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H Merged image. The competence to produce outgrowths after 7 days in culture was significantly decreased in the three groups of cloned embryos when compared with both control fertilized and parthenogenetic embryos, and significant differences were also observed between the ME and the AE cloned groups. In spite of this, efficiencies of ESC lines derivation, ranging from 9. A total of two-cell stage cloned embryos generated from cytoplasts prepared by ME were transferred into the oviducts of 12 surrogate females and, as a result, two cloned pups developed to term and were born alive Table 5.

Full-term development was also obtained, for the first time in the mouse, using cytoplasts prepared by AE procedures. In this case, from the cloned embryos transferred into 14 females, one stillborn and one cloned pup born alive were obtained. No full-term development resulted from the cloned embryos produced with IE cytoplasts transferred into the oviducts of 13 females.

Table 5 Comparative full-term development of control in vivo fertilized embryos and of cloned embryos produced from enucleated oocytes prepared by mechanical ME , assisted AE , or induced IE enucleation procedures. All cloned pups that were born alive had body weights equivalent to those of non-cloned pups Table 5. The placenta weights of the clones obtained with ME and AE procedures were very similar, but between two and three times heavier than those of the noncloned pups produced from in vivo fertilized embryos.

Successful development of cloned embryos requires the efficient reprogramming of the donor nucleus in order to silence somatic gene expression and to activate an embryonic pattern of gene expression.


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In addition, as nuclear reprogramming in cloned embryos is thought to occur at an epigenetic level and as one of the epigenetic pathways related to chromatin structure is the global level of histone acetylation Yang et al. In contrast, in IE cytoplasts, the deacetylation of H3K14 occurred at a much lower level and re-acetylation started before a complete deacetylation had been reached. Alternatively, the two-step activation protocol used in the IE group could also account for the differences observed with the ME and AE groups, in which a single step of SrCl 2 activation was applied.

Whether the dynamics of H3K14 acetylation observed in ME and AE groups corresponds to a more or less efficient nuclear reprogramming compared with IE cytoplasts, however, is not clear. On the one hand, it has been suggested that hyperacetylation of core histones is correlated with a transcriptional permissive state of the chromatin. This is thought to be necessary for the activation of embryonic genes during nucleus reprogramming and could explain the beneficial effects of applying HDACis treatments before and during activation in cloned embryos produced with ME procedures to prevent histone deacetylation in the transferred nucleus Rybouchkin et al.

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In this sense, the lower deacetylation activity in the IE cytoplasts could represent an advantage of the IE protocol with regard to ME or AE methods concerning nuclear reprogramming. But, on the other hand, studies indicate that a complete deacetylation of the lysine residues of core histones before the beginning of the re-acetylation, as occurred in our ME and AE cytoplasts, may be necessary for the correct regulation of gene expression and the establishment of totipotency in the cloned embryos Wang et al.

Histone deacetylation would allow the silencing of the somatic gene expression program before the initiation of the embryonic pattern of gene expression is induced by histone re-acetylation.


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This acetylation dynamic is probably facilitated by the remodeling of the transferred nucleus, including nuclear envelope breakdown and premature chromosome condensation, which in MII oocytes are promoted by the activities of maturation promoting factor MPF and mitogen-activated protein kinases MAPK; Kono In our IE protocol, oocytes are activated immediately after NT, resulting in a quick decline in MPF and MAPK levels that could limit the time over which nuclear remodeling occurs and allow the expression of embryonic genes before the somatic gene expression program has been turned off.

Which of the two histone acetylation patterns observed in our study corresponds with a higher reprogramming capacity of the cytoplasts was not reflected in the in vitro development rates of the cloned embryos or in the quality of the blastocysts produced. Thus, although the rates of development to the two-cell and morula stages were lower in the IE group, the final frequencies of blastocyst formation were equivalent to those obtained in the ME group, indicating a similar reprogramming capacity between the two types of cytoplasts.


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These results are in accordance to our previous findings, showing that injection of the donor nucleus into IE cytoplasts prior to the activation and the antimitotic treatment results in similar blastocyst rates than when using ME procedures Costa-Borges et al.

Furthermore, results of this study also show that the quality of the SCNT blastocysts produced, in terms of total and ICM mean cell numbers, is not affected by the enucleation method applied. Differences in H3K14 acetylation dynamics were neither translated to the efficiencies of ntESC derivation from the three groups of blastocysts. Outgrowth formation was less efficient in cloned embryos than in control fertilized or parthenogenetically activated blastocysts and, because parthenogenetic blastocysts showed a higher total mean number of cells than all groups of cloned blastocysts, these differences could be explained by their higher proliferation capacity.

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Nevertheless, production of ESC lines after extensive culture of the outgrowths was similarly efficient in all groups of embryos, and all ntESC and control ESC lines displayed an equivalent morphology of the colonies and an equivalent pattern of expression of pluripotency markers. The observed efficiencies of ESC derivation from our control embryos are similar to those previously reported by Gong et al. In line with these previous studies, it is, therefore, not surprising that VPA-treated cloned blastocysts derived from ME procedures in this study show the same potential as control fertilized and parthenogenetic embryos to produce ESC lines.

And, according to our results, this potential would not be affected by the enucleation protocol used to produce the cloned embryos. Compared with preimplantation development and ntESC derivation, development to term is much more constrained by genetic and epigenetic abnormalities of the embryos. As a result, the potential of cloned embryos to develop into live pups is much lower than to develop into blastocysts or to produce ntESC lines Yang et al.

For this reason, in this study, we also wanted to determine the potential of the SCNT embryos produced to develop to term after transferring them into surrogate females. For the first time, a cloned mouse was successfully obtained from cloned embryos prepared by AE. Healthy cloned animals from different species, including pigs, cows, cats, and goats, have previously been produced by others using AE procedures, with efficiencies equivalent to those obtained using mechanically enucleated oocytes Yin et al.

Similarly, in our study, the cloning efficiency using AE cytoplasts was equivalent to that of ME cytoplasts, demonstrating that AE cytoplasts are competent to fully reprogram the transferred nucleus in the presence of VPA and, therefore, that the AE procedure can be used as an alternative method to ME to enucleate mouse oocytes. The discrepancy of results in the mouse species can be probably attributed to strain-specific differences, as not all HDACis are equally effective in all genetic backgrounds Kishigami et al. With regard to the IE procedure, although Gasparrini et al.

Even though a much higher number of embryos should be transferred to reach a conclusion, due to the low cloning efficiency in general with the mouse strain used in this study, the lack of offspring from cloned embryos produced from IE cytoplasts apparently indicates a reduced reprogramming capacity of IE cytoplasts when compared with that of ME or AE cytoplasts. And, according to the particular H3K14 acetylation dynamics of the donor nucleus observed in this group of cloned embryos, this could suggest that histone deacetylation may be required before activation-induced re-acetylation, to achieve a correct pattern of gene expression in the cloned embryo that allows full-term development.

Because nuclear reprogramming is a slow and progressive process and because events that occur early after nucleus injection may be essential prerequisites for later events Latham , the extent of nuclear reprogramming achieved in cloned IE embryos could have been sufficient for the dedifferentiation of the somatic nucleus to a totipotent embryonic state, but insufficient for the redifferentiation to different somatic cell types during post-implantation development Yang et al.

Nuclear Transfer in the sheep; William A. Ritchie; 3. Boquest, Aboulghassem Shahdadfar, Jan E. Brinchmann and Philippe Collas; 5. Broyles, Austin C. Roth, Mairead Todd and Visar Belegu; 6. Epigenetic reprogramming of somatic genomes by electrofusion with embryonic stem cells; Masako Tada and Takashi Tada; 8. Gaustad and Philippe Collas; Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos; Fatima Santos and Wendy Dean; Terry and David M. Gilbert; Allan King; Wobus; Fenderson, Maria P.

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Conservation Land Management. Go to Conservation Land Management. Series: Methods in Molecular Biology Volume: Publisher: Humana Press. Click to have a closer look. Select version. About this book Contents Customer reviews Related titles. Images Additional images. About this book A wide-ranging collection of readily reproducible methods for performing nuclear reprogramming by nuclear transfer in several different species, by fusion through both chemical treatment and electrically shocking cells, and by in vivo treatment of cells with cell extracts.