Project description:Improving protein production in human embryonic kidney cells is important structural studies and antibody production. The use of small non-protein coding RNA, such as microRNA, has been an effective method for increasing protein expression. Our high-throughput human microRNA screen in HEK 293 cells previously identified miRNA 22-3p as a promising candidate for increasing the expression of luciferase, couple of membrane proteins and a secreted fusion protein. To explore the mechanisms of this increase in protein expression and to understand the intracellular events, we conducted a gene expression analysis of luciferase- expressing HEK 293 cells transfected with a mir-22-3p mimic and compared with cells transfected with a negative control. Following microarray analysis, down-regulated genes were identified and were cross-referenced with the predicted targets of mir-22-3p and with results from our previous high-throughput siRNA screen. By performing common seed analysis on the possible targets, the list was narrowed to two genes, HIPK1 and FRAT2. These two genes were validated as being involved in improving luciferase production using siRNA and qRT PCR.
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:Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. We found significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells Comparison of three HEK-293 suspension cell lines transcriptomics during an AAV production process
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:Comparison of the gene expression profiles of a recombinant protein producing Hek 293 cell line (referred to as producer) and its non-producing parental cell line Hek293F (referred to as non-producer). The parental cell line was obtained from Invitrogen, Carlsbad, CA. The producer was transfected with a heavy chain variable region fused to the Fc region of a human IgG (dAb-Fc). The aim of this study was to gain a better understanding of the process of recombinant protein production in Hek293 cells and to identify targets for the engineering of an improved host cell line.
2012-12-20 | GSE36094 | GEO
Project description:HEK 293 T cell RNA-seq by overexpressing mir-199a-3p
Project description:Schizophrenia-associated miRNA were bidirectionally modulated in HEK-293, HeLa, and SH-SY5Y cell models. Results provide important insights into the current understanding of miRNA function in various cellular environments. Total RNA was obtained from HEK-293, HeLa, and SH-SY5Y cells at 24hrs post-transfection with either synthetic miRNA (miR overexpression) or anti-miR inhibitor (miR inhibition) oligonucleotides.
Project description:Oxaliplatin (oxPt) resistance in colorectal cancers (CRC) is a major unsolved problem. Consequently, predictive markers and a better understanding of resistance mechanisms are urgently needed. To investigate if the recently identified predictive miR-625-3p is functionally involved in oxPt resistance, stable and inducible models of miR-625-3p dysregulation were analyzed. Ectopic expression of miR-625-3p in CRC cells led to increased resistance towards oxPt. The mitogen-activated protein kinase (MAPK) kinase 6 (MAP2K6/MKK6) – an activator of p38 MAPK - was identified as a functional target of miR-625-3p, and, in agreement, was down-regulated in patients not responding to oxPt therapy. The miR-625-3p resistance phenotype could be reversed by anti-miR-625-3p treatment and by ectopic expression of a miR-625-3p insensitive MAP2K6 variant. Transcriptome, proteome and phosphoproteome profiles revealed inactivation of MAP2K6-p38 signaling as a possible driving force behind oxPt resistance. We conclude that miR-625-3p induces oxPt resistance by abrogating MAP2K6-p38 regulated apoptosis and cell cycle control networks.
Project description:MicroRNAs can play important roles in gene regulation affecting both normal development and diseases, including cancer. This microarray experiment was performed to identify genes and pathways differentially expressed in MOLT4 T-leukemia cells engineered to overexpress hsa-pre-miR-22-3p. MOLT4 cells transduced with an empty or hsa-pre-miR-22-3p lentiviral vectors were collected, processed for RNA extraction and hybridized on Affymetrix Clariom™ S Human arrays (n=3 replicates/group). Raw microarray data are available together with the applied protocols.
Project description:Comparison of the gene expression profiles of a recombinant protein producing Hek 293 cell line (referred to as producer) and its non-producing parental cell line Hek293F (referred to as non-producer). The parental cell line was obtained from Invitrogen, Carlsbad, CA. The producer was transfected with a heavy chain variable region fused to the Fc region of a human IgG (dAb-Fc). The aim of this study was to gain a better understanding of the process of recombinant protein production in Hek293 cells and to identify targets for the engineering of an improved host cell line. Duplicate samples for RNA extraction were taken at four time points (approximately 30, 43, 51 and 67 hours after inoculation of the bioreactor) during the exponential phase of batch bioreactor cultures. Only one of these technical duplicates was analysed. Three bioreactor cultures were performed for each cell line representing three biological replicates resulting in 12 (3 x 4) samples per cell line.