Mechanical regulation of the electric maturation of hippocampal neurons
Ontology highlight
ABSTRACT: In this project, we cultured rat hippocampal neurons in mechanically different environments and studied electrical maturation. We compared WT neurons with two different Piezo1 knockdown conditions. These two knockdown conditions were generated in two independent CRISPR-Cas9 KD assays. Each assay is based on four different CRISPR-Cas9 guides targeting the Piezo1 gene. RNA sequencing was used to analyse the pathway leading to the stiffness dependent maturation behaviour.
Project description:The expression profile in miR-155-/- FLT3-ITD+ AML is unknown. Using empty vector (EV) or two distinct miR-155 (S3 or S10) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing to determine the expression profile in these cells dependent on miR-155. We found a number of pathways dysregulated, including STAT5 activation. RNAseq was performed on EV or miR-155 lentiviral CRISPR-Cas9 infected MV4-11 cell lines in triplicate cultures.
Project description:The expression level of microRNAs in FLT3-ITD+ AML is unknown. Using empty vector (EV) lentiviral CRISPR-Cas9 infected FLT3-ITD+ AML cell lines (MV4-11 cells), we performed next generation RNA sequencing on small RNAs to determine microRNA expression level in these cells. We found a variety of evolutionarily conserved and non-conserved microRNAs expressed in our cells of interest. Small RNAseq on EV lentiviral CRISPR-Cas9 infected MV4-11 cell lines was performed on triplicate cultures.
Project description:The functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to test the impact of individual microRNAs on the growth of FLT3-ITD positive leukemia cells. This approach identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote tumor cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. Our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease. Loss-of-function CRISPR-Cas9 screen identifies genes whose loss leads to increased or decreased FLT3-ITD+ cell growth over 23 day time-course
Project description:We generated these data to compare two modifications of the original ATAC-seq protocol. One was the cleavage of mtDNA using CRISPR/Cas9 and 100 gRNAs targeting mtDNA. The other was the removal of detergent from the cell lysis step. There are 27 sample pairs, untreated and treated with anti-mt CRISPR/Cas9 grouped by sample pair number. Refer to Supplemental File 1 of the article describing this data set for more information on the samples.
Project description:The specific functional features of the epidermal keratinocytes are determined by the activity of many genes. The aim of the project was to characterize the role of HSPA2, a member of the HSPA chaperone family (HSP70), in human epidermal keratinocytes. The inactivation of the HSPA2 gene in the HaCaT line of spontaneously immortalized epidermal keratinocytes was performed by CRISPR/Cas9 gene editing system. Next, the effect of modifications on the transcriptomic profile of cells growing in 2D monolayer culture was investigated. This study was supported by a Polish National Science Center grant number NCN 2017/25/B/NZ4/01550.
Project description:The specific functional features of the epidermal keratinocytes are determined by the activity of many genes. The aim of the project was to characterize the role of HSPA2, a member of the HSPA chaperone family (HSP70), in human epidermal keratinocytes. The inactivation of the HSPA2 gene in the HaCaT line of spontaneously immortalized epidermal keratinocytes was performed by CRISPR/Cas9 gene editing system. Next, the effect of modifications on the transcriptomic profile of cells growing in a three-dimensional model of reconstructed human epidermis in vitro was investigated. The cells were grown at air-liquid interface culture on collagen-fibroblast matrix to achieve maximal level of HaCaT differentiation in RHE system.
Project description:Since LAMC2 is a secreted molecule present in the extracellular matrix of the cells, was designed a strategy based on CRISPR/Cas9-mediated homologous recombination to mark LAMC2 cells in human PDACs. A Cas9 single-guide RNAs complementary to sequences overlapping the stop codon of the LAMC2 locus was designed and a donor vector that contained LAMC2 homology arms flanking an EGFP reporter cassette positioned immediately upstream of the stop codon was generated. LF2A self-cleavage peptide in frame with EGFP so that LAMC2-EGFP locus was expressed as a single mRNA was added, whereas the resulting polypeptide was cleaved in the two encoded proteins, LAMC2 and EGFP. L3.6pl and PANC-1 cells were nucleofected with the donor vector together with a guide-RNA-Cas9 (guide). The engineered cells were subcutaneously injected in CD1 male mice and RNA-seq was performed on LAMC2-EGFP+ and EGFP- -derived tumors.
Project description:Summary: Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However, the entire biological role for Rnh1 is unknown. We generated RNH1 knock out K562 cells by CRISPR/Cas9 method. Here we studied differential gene expression from wild type and RNH1 knock out K562 cells by RNA-Seq analysis. Overall design: Total RNA was isolated from wild type and RNH1 deficient K562 cells.
Project description:CRISPR/Cas9 system was used to generate mediator complex subunit 1 (MED1) knockout human pre-B ALL cell line 697. RNA-seq was performed to observe the effects of MED1 deletion on gene expression in 697.