Project description:HEK-293-derived cell lines depleted of Dicer or one of four Argonaute proteins were generated. Microarray analysis of these knock down cell lines and various control cell lines was performed to address how large the proportion of genes is that are regulated by RNA silencing in an individual cell type. Comparison of different arrays revealed commonly (up) regulated genes. The highest overlap was observed in Ago2 and Dicer knockdown cells. Experiment Overall Design: three control cell lines (T-Rex, shRNA, empty vector), two induvidual Argonaute 1 and 4 (Ago1kd, Ago4kd) knock down cell lines, one Argonaute 2 and 3 (Ago2kd, Ago3kd) and two induvidual Dicer knock down cell lines were analyzed as biological replicates
Project description:HEK-293-derived cell lines depleted of Dicer or one of four Argonaute proteins were generated. Microarray analysis of these knock down cell lines and various control cell lines was performed to address how large the proportion of genes is that are regulated by RNA silencing in an individual cell type. Comparison of different arrays revealed commonly (up) regulated genes. The highest overlap was observed in Ago2 and Dicer knockdown cells. Keywords: different timepoints of induction of stable knock down cell lines compared to various control cell lines
Project description:Dicer is a central enzymatic player in RNA interference (RNAi) pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, there is little known about any possible nuclear function. Here we show that Dicer is present in both the nucleus and cytoplasm, but that its nuclear levels are tightly regulated. In its nuclear manifestation Dicer interacts with RNA polymerase II (Pol II) at actively-transcribed gene loci. Loss of Dicer causes the appearance of endogenous dsRNA, leading to induction of the interferon response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping non-coding RNA transcription units. Failure to do so has catastrophic effects on cell function. Taken together, we present here a micro (mi)RNA independent role for human nuclear Dicer. DICER ChIP-seq profile in wt 293 HEK cells, and dsRNA-seq profile in wt and DICER-depleted 293 HEK cells
Project description:The aim of this study is to discover genes regulated by miR-204. Differential gene expression in HEK-293 cells transfected with miR-204-mimic compared to HEK-293 cells transfected with control oligo (HEK-293 control) was analyzed using the Agilent Human Whole Genome 4x44K gene expression array (Agilent Technologies, Santa Clara, CA).
Project description:The aim of this study is to discover genes regulated by miR-204. Differential gene expression in HEK-293 cells transfected with miR-204-mimic compared to HEK-293 cells transfected with control oligo (HEK-293 control) was analyzed using the Agilent Human Whole Genome 4x44K gene expression array (Agilent Technologies, Santa Clara, CA). HEK-293 cells were transfected with either miR-204 or a control, and gene expression was analyzed using the Agilent Human Whole Genome 4x44K array. A dye-swap was performed.
Project description:To determine the target genes of RBM10,we have employed microarray based gene expression profiling by knocking down RBM10 in HEK 293 cells. Microarray analysis after RBM10 knockdown on HEK 293 cells showed that over 1000 genes were down regulated while another 800 genes up regulated as a result of the knockdown. Among the down regulated genes, we found the significant presence of cardiovascular disease related genes, especially cardiac hypertrophy and heart failure.
Project description:To obtain evidence that Argonaute (AGO) crosslink-centered regions (CCRs) indeed contain functional miRNA-binding sites, we blocked 25 of the most abundant miRNAs in HEK 293 cells (Figure 5C of PMID 20371350) by transfection of a cocktail of 2'-O-methyl-modified antisense oligoribonucleotides and monitored the changes in mRNA stability by microarrays (Figure 7A of PMID 20371350). Consistent with previous studies of individual miRNAs (Grimson et al., 2007), the magnitude of the destabilization effects of transcripts containing at least one CCR depended on the length of the seed-complementary region and dropped from 9-mer to 8-mer to 7-mer to 6-mer matches (Figure 7B of PMID 20371350). We did not find evidence for significant destabilization of transcripts that only contained imperfectly paired seed regions. The top 25 expressed miRNAs expressed in HEK 293 cells were inhibited by transfection of a cocktail of 25 antisense 2'-O-Me oligoribonucleotides and the gene expression was compared to mock-transfected cells.
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Initially, we will determine gene expression patterns in human cells embryonic kidney cells (HEK 293 cell line) overexpressing different isoforms of hIMP1 protein. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). We will examine in HEK 293 cells effect of over expression of 1) hIMP1 wt protein, 2) over expression of dominant negative isoform of hIMP1 protein and compare their gene expression patterns to 3) HEK 293 cells treated with mock vector. HEK 293 cells were seeded on 10 cm plates and transfected next day using Lipofectamine Plus reagent. 24 hours after cells were washed twice with ice cold PBS buffer and lysed with TRizol reagent directly on plates. Total RNA was extracted, purified according the manufacturer protocol and stored at -800C. We will provide 2 RNA samples for each hIMP1 isoform used in two different transfection experiment to reduce any spurious expression differences resulting from culture condition or transfection efficiency variations. Keywords: dose response
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Initially, we will determine gene expression patterns in human cells embryonic kidney cells (HEK 293 cell line) overexpressing different isoforms of hIMP1 protein. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). We will examine in HEK 293 cells effect of over expression of 1) hIMP1 wt protein, 2) over expression of dominant negative isoform of hIMP1 protein and compare their gene expression patterns to 3) HEK 293 cells treated with mock vector. HEK 293 cells were seeded on 10 cm plates and transfected next day using Lipofectamine Plus reagent. 24 hours after cells were washed twice with ice cold PBS buffer and lysed with TRizol reagent directly on plates. Total RNA was extracted, purified according the manufacturer protocol and stored at -800C. We will provide 2 RNA samples for each hIMP1 isoform used in two different transfection experiment to reduce any spurious expression differences resulting from culture condition or transfection efficiency variations.