Ontology highlight
ABSTRACT:
PROVIDER: PRJNA633843 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11819090.fastq.gz | Fastqsanger.gz | |||
| SRR11819091.fastq.gz | Fastqsanger.gz | |||
| SRR11819092.fastq.gz | Fastqsanger.gz | |||
| SRR11819093.fastq.gz | Fastqsanger.gz | |||
| SRR11819094.fastq.gz | Fastqsanger.gz |
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Project description:With the recent advancements in genome editing, next generation sequencing (NGS), and scalable cloning techniques, scientists can now conduct genetic screens at unprecedented levels of scale and precision. With such a multitude of technologies, there is a need for a simple yet comprehensive pipeline to enable systematic mammalian genetic screening. In this study, we develop novel algorithms for target identi fication and a toxin-less Gateway cloning tool, termed MegaGate, for library cloning which, when combined with existing genetic perturbation methods and NGS-coupled readouts, enable versatile engineering of relevant mammalian cell lines. Our integrated pipeline for Sequencing-based Target Ascertainment and Modular Perturbation Screening (STAMPScreen) can thus be utilized for a host of cell state engineering applications.
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Project description:Animal cloning can be achieved through somatic cell nuclear transfer (SCNT), yet the success rate is very low. Recent studies have revealed H3K9me3 in donor cells and abnormal Xist activation as epigenetic barriers that impede SCNT reprogramming. Here we overcome both barriers by using Xist knockout donor cells combined with overexpressing Kdm4d and achieved the highest cloning efficiency in mice. However, post-implantation developmental defects and abnormal placenta were still observed, indicating presence of additional epigenetic barriers impedes SCNT cloning. Comparative DNA methylome analysis of IVF and SCNT blastocysts identified many abnormally methylated regions in SCNT embryos, despite successful global methylome reprogramming. Strikingly, allelic transcriptome and ChIP-seq analyses of preimplantation SCNT embryos revealed a complete loss of H3K27me3 imprinting, which likely accounts for postimplantation developmental defects of SCNT embryos. This study not only provides an efficient method for mouse cloning, but also paves the way for further improving SCNT cloning efficiency.
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Project description:Since the creation of Dolly, the first sheep cloned via somatic cell nuclear transfer (SCNT), in 1997, more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1-5%) is that the clones are likely derived from adult stem cells, which form an extremely small fraction in most adult tissues. Support for this hypothesis is that the cloning efficiency of full term development using embryonic stem (ES) cells as nuclear donors is 5-10 times higher than that for somatic cells as nuclear donors. Additionally, cloned pups could not be produced directly from cloned embryos derived from nuclei of differentiated B and T cells or neuronal cells. The question remains: can SCNT-derived animal clones be derived from truly differentiated somatic cells? We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells (HSCs), progenitor cells (HPCs), and granulocytes. Surprisingly, we found that cloning efficiency increases over the differentiation hierarchy. The terminally differentiated post-mitotic granulocytes yielded the greatest cloning efficiency and we produced two cloned pups from granulocytes. We conclude that cloned mammals could be directly derived from post-mitotic differentiated somatic cells. Keywords: cell type comparison
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Project description:In this study, the DEGs in leaves of R. soongorica in response to PEG-induced drought stress, UV-B radiation, combined stress by UV-B radiation and PEG-induced drought, and combined stresses by UV-B radiation, PEG-induced drought and NaCl in contrast to control group were examined, respectively, using DGE tag profiling technology. Analysis of gene expression related to stress response should provide further insight into the molecular mechanisms of stress tolerance in R. soongorica. Based on the putative functions of the identified genes, some important genes may be cloned. Moreover, the cloning of stress tolerance genes and determination of their expression patterns may offer some attractive candidate genes and valuable information for improving stress tolerance of plants through genetic engineering.
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Project description:Since the creation of Dolly, the first sheep cloned via somatic cell nuclear transfer (SCNT), in 1997, more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1-5%) is that the clones are likely derived from adult stem cells, which form an extremely small fraction in most adult tissues. Support for this hypothesis is that the cloning efficiency of full term development using embryonic stem (ES) cells as nuclear donors is 5-10 times higher than that for somatic cells as nuclear donors. Additionally, cloned pups could not be produced directly from cloned embryos derived from nuclei of differentiated B and T cells or neuronal cells. The question remains: can SCNT-derived animal clones be derived from truly differentiated somatic cells? We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells (HSCs), progenitor cells (HPCs), and granulocytes. Surprisingly, we found that cloning efficiency increases over the differentiation hierarchy. The terminally differentiated post-mitotic granulocytes yielded the greatest cloning efficiency and we produced two cloned pups from granulocytes. We conclude that cloned mammals could be directly derived from post-mitotic differentiated somatic cells. Experiment Overall Design: single channel Affymetrix arrays to confirm cell-type specific gene expression profiles
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Project description:Somatic embryos are very much similar to zygotic counterparts in many morphological aspects and the somatic embryos are derived from somatic cells by undergoing various metabolic regulations. The somatic embryos have been used in artificial seed technology, genetic engineering and germplasm conservation. Though somatic embryo development is an important topic in growth and developmental studies, the molecular mechanism underlying the developmental process remains unclear. Therefore, understanding the molecular basis behind somatic embryo development can provide insight on the signaling pathways integrating this process. Proteomic analysis of somatic embryo development in cv. Grand Naine (AAA) was carried out to identify the differentially accumulated protein using two dimensional gel electrophoresis coupled with mass spectrometry. In total, 25 protein spots were differentially accumulated in different developmental stages of somatic embryos. Among them, three proteins were uniquely present in 30 days globular stage somatic embryos and six proteins were uniquely present in 60 days matured somatic embryo. Functional annotation of identified spots showed that major proteins are involved in growth and developmental process (17 %) followed by defense response (12%) and signal transportation events (12 %). In early stage, cell division and growth related proteins were involved in the induction of somatic embryos whereas in late developmental stage, cell wall modification proteins along with stress related proteins like played a defense role against dehydration and osmotic stress and resulted in maturation of somatic embryo. Alongside some identified stage specific proteins are valuable indicators and have been used as genetic markers.
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