scholarly journals High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Shengtai Bian ◽  
Yicen Zhou ◽  
Yawei Hu ◽  
Jing Cheng ◽  
Xiaofang Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Cell Electroporation ◽  
Guide Rnas

The Role of Sprouty Family Members in Hematopiesis in Zebrafish and Mammals.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 137-137
Author(s):  
Eric M. Mendenhall ◽  
Craig E. Eckfeldt ◽  
Stephen C. Ekker ◽  
Catherine M. Verfaillie
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
High Throughput ◽  
Mammalian Cells ◽  
Mapk Pathway ◽  
Family Members ◽  
Gene Array ◽  
Hematopoietic Stem

Abstract In an effort to identify novel regulators of hematopoiesis, we identified a Sprouty family member, SPRY1, significantly enriched (>1.5 fold, p-value < 0.03) in human hematopoietic stem cells (HSC) compared to more committed hematopoietic progenitor cells (HPC) using Affymetrix-based global gene expression profiling (see Eckfeldt et al. abstract). Predominantly FGF, but also VEGF, and EGF signaling induces expression of Sprouty family members (SPRY1-4). SPRY proteins form heterodimers with other Sprouty members along with homodimers. They and are intracellular antagonists and agonists of receptor tyrosine kinase signaling via the RAS/MAPK pathway. SPRY proteins are best characterized as antagonists of the FGF, EGF, and VEGF pathways. As part of a high-throughput functional analysis of the genes identified by human gene array in zebrafish, we evaluated the role of the only identified zebrafish homologue for Sprouty, SPRY4, in zebrafish hematopoiesis. Using morpholino (MO) antisense technology we knocked-down expression of zebrafish SPRY4 during early zebrafish development in a double transgenic GATA1:dsRed/Fli1:eGFP zebrafish line. The SPRY4 morpholino injected fish (SPRY4[superscript]MO[/superscript]) showed an almost complete loss of blood with normal vasculature, which was confirmed by injection of a second unique morpholino designed against SPRY4. [italics] In situ [/italics]hybridization of the hematopoietic markers SCL, GATA1 and Ikaros were all significantly reduced or absent at multiple developmental stages in the SPRY4[superscript]MO[/superscript] embryo. Together with the normal development of the vasculature, this data indicates SPRY4 acts early in hematopoiesis and after the specification of the hemangioblast. [italics] In situ[/italics] hybridization for SPRY4 demonstrated expression in the otic vesicle, tail bud and lateral plate mesoderm, the first site of hematopoietic stem cells. Coinjection of SPRY morpholino and low doses of VEGF morpholino did not rescue the SPRY blood phenotype, indicating that the blood phenotype is not likely through the VEGF pathway. Previous gain-of-function experiments injecting SPRY4 mRNA showed an expansion of hematopoietic progenitors (Furthauer et al. Development 2001), a phenotype we confirmed with overexpression of SPRY4 cDNA. Experiments are ongoing to overexpress human SPRY1 or zebrafish SPRY4 cDNAs in mammalian cells to determine if the hematopoietic phenotype correlates with the zebrafish data. Additional genes differentially expressed in human HSC and HPC evaluated via the high throughput zebrafish screen will also be presented. This is the first description of functional validation of a gene from a human microarray in zebrafish, an essential step in obtaining meaningful in vivo data from global gene profiling studies.

scholarly journals Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Zhang ◽  
Tuan M. Nguyen ◽  
Xiao-Ou Zhang ◽  
Limei Wang ◽  
Tin Phan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput ◽  
Biological Effect ◽  
Human Hepatocellular Carcinoma ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Screening Library ◽  
High Throughput Study

AbstractShort hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

High-throughput cocrystal slurry screening by use of in situ Raman microscopy and multi-well plate

2010 ◽  
Vol 399 (1-2) ◽  
pp. 52-59 ◽  
Author(s):  
Takashi Kojima ◽  
Shunichirou Tsutsumi ◽  
Katsuhiko Yamamoto ◽  
Yukihiro Ikeda ◽  
Toshiya Moriwaki
Keyword(s):  
High Throughput ◽  
Raman Microscopy ◽  
In Situ Raman

scholarly journals Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

2016 ◽  
Vol 82 (15) ◽  
pp. 4757-4766 ◽  
Author(s):  
Caterina R. Giner ◽  
Irene Forn ◽  
Sarah Romac ◽  
Ramiro Logares ◽  
Colomban de Vargas ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
Relative Abundance ◽  
Ribosomal Dna ◽  
18S Rdna ◽  
High Throughput Sequencing ◽  
454 Sequencing ◽  
Epifluorescence Microscopy ◽  
Environmental Sequencing

ABSTRACTHigh-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescencein situhybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances.IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescencein situhybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.

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