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Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions.

ABSTRACT: Gel-based artificial organelles have been developed that enable sequence-specific and programmable localization of cell-free transcription and translation reactions inside an artificial cellular system. To this end, we utilize agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. We show that RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. We also demonstrate that mRNA molecules, produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles containing their cognate DNA triggers. Spatial confinement of transcription and translation in separate organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.

SUBMITTER: Aufinger L

PROVIDER: S-EPMC6640049 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions.

Aufinger Lukas L Simmel Friedrich C FC

Angewandte Chemie (International ed. in English) 20181127 52

Gel-based artificial organelles have been developed that enable sequence-specific and programmable localization of cell-free transcription and translation reactions inside an artificial cellular system. To this end, we utilize agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. We show that RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the ...[more]

PMID: 30394633

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Project description:BACKGROUND:During the preimplantation phase in the pig, the conceptus trophoblast elongates into a filamentous form and secretes estrogens, interleukin 1 beta 2, interferons, and other signaling molecules before attaching to the uterine epithelium. The processes in the uterine endometrium in response to conceptus signaling are complex. Thus, the objective of this study was to characterize transcriptome changes in porcine endometrium during the time of conceptus attachment considering the specific localization in different endometrial cell types. RESULTS:Low-input RNA-sequencing was conducted for the main endometrial compartments, luminal epithelium (LE), glandular epithelium (GE), blood vessels (BV), and stroma. Samples were isolated from endometria collected on Day 14 of pregnancy and the estrous cycle (each group n = 4) by laser capture microdissection. The expression of 12,000, 11,903, 11,094, and 11,933 genes was detectable in LE, GE, BV, and stroma, respectively. Differential expression analysis was performed between the pregnant and cyclic group for each cell type as well as for a corresponding dataset for complete endometrium tissue samples. The highest number of differentially expressed genes (DEGs) was found for LE (1410) compared to GE, BV, and stroma (800, 1216, and 384). For the complete tissue, 3262 DEGs were obtained. The DEGs were assigned to Gene Ontology (GO) terms to find overrepresented functional categories and pathways specific for the individual endometrial compartments. GO classification revealed that DEGs in LE were involved in 'biosynthetic processes', 'related to ion transport', and 'apoptotic processes', whereas 'cell migration', 'cell growth', 'signaling', and 'metabolic/biosynthetic processes' categories were enriched for GE. For blood vessels, categories such as 'focal adhesion', 'actin cytoskeleton', 'cell junction', 'cell differentiation and development' were found as overrepresented, while for stromal samples, most DEGs were assigned to 'extracellular matrix', 'gap junction', and 'ER to Golgi vesicles'. CONCLUSIONS:The localization of differential gene expression to different endometrial cell types provided a significantly improved view on the regulation of biological processes involved in conceptus implantation, such as the control of uterine fluid secretion, trophoblast attachment, growth regulation by Wnt signaling and other signaling pathways, as well as the modulation of the maternal immune system.

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Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions.

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Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions.

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