scholarly journals Prion protein (PrP) gene-knockout cell lines: insight into functions of the PrP

2015 ◽  
Vol 2 ◽  
Author(s):  
Akikazu Sakudo ◽  
Takashi Onodera
Keyword(s):  
Prion Protein ◽  
Cell Lines ◽  
Gene Knockout ◽  
Prp Gene ◽  
Insight Into

scholarly journals Monosomy 3 Is Linked to Resistance to MEK Inhibitors in Uveal Melanoma

2021 ◽  
Vol 22 (13) ◽  
pp. 6727
Author(s):  
Svenja Mergener ◽  
Jens T. Siveke ◽  
Samuel Peña-Llopis
Keyword(s):  
Cell Lines ◽  
Uveal Melanoma ◽  
Survival Data ◽  
Translation Initiation Factor ◽  
The Cancer Genome Atlas ◽  
Initiation Factor ◽  
Chromosome 3 ◽  
Mek Inhibitors ◽  
Mek Inhibition ◽  
Insight Into

The use of MEK inhibitors in the therapy of uveal melanoma (UM) has been investigated widely but has failed to show benefits in clinical trials due to fast acquisition of resistance. In this study, we investigated a variety of therapeutic compounds in primary-derived uveal melanoma cell lines and found monosomy of chromosome 3 (M3) and mutations in BAP1 to be associated with higher resistance to MEK inhibition. However, reconstitution of BAP1 in a BAP1-deficient UM cell line was unable to restore sensitivity to MEK inhibition. We then compared UM tumors from The Cancer Genome Atlas (TCGA) with mutations in BAP1 with tumors with wild-type BAP1. Principal component analysis (PCA) clearly differentiated both groups of tumors, which displayed disparate overall and progression-free survival data. Further analysis provided insight into differential expression of genes involved in signaling pathways, suggesting that the downregulation of the eukaryotic translation initiation factor 2A (EIF2A) observed in UM tumors with BAP1 mutations and M3 UM cell lines might lead to a decrease in ribosome biogenesis while inducing an adaptive response to stress. Taken together, our study links loss of chromosome 3 with decreased sensitivity to MEK inhibition and gives insight into possible related mechanisms, whose understanding is fundamental to overcome resistance in this aggressive tumor.

scholarly journals Prion Protein Gene-Deficient Cell Lines: Powerful Tools for Prion Biology

2007 ◽  
Vol 51 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Akikazu Sakudo ◽  
Takashi Onodera ◽  
Kazuyoshi Ikuta
Keyword(s):  
Prion Protein ◽  
Cell Lines ◽  
Protein Gene ◽  
Prion Protein Gene ◽  
Deficient Cell

scholarly journals Prion protein gene polymorphisms in healthy and scrapie-affected sheep in Greece

2004 ◽  
Vol 85 (2) ◽  
pp. 547-554 ◽  
Author(s):  
Charalambos Billinis ◽  
Vassilios Psychas ◽  
Leonidas Leontides ◽  
Vassiliki Spyrou ◽  
Stamatis Argyroudis ◽  
...  
Keyword(s):  
Prion Protein ◽  
Protein Gene ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Gradient Gel Electrophoresis ◽  
Healthy Control ◽  
The Moment ◽  
Chios Sheep ◽  
Prp Gene ◽  
Control Study ◽  
Clinical Cases

A total of 216 local crossbred sheep from 16 scrapie-affected Greek flocks and 210 purebred sheep of the milk breeds Chios and Karagouniko from healthy flocks were analysed for scrapie-linked polymorphisms in the prion protein (PrP) gene. Of the 216 sheep in this case–control study, 96 sheep were clinical cases, 25 subclinical cases (asymptomatic at the moment of euthanasia but positive by histopathology and/or ELISA detecting proteinase-resistant PrP) and 95 healthy controls (negative by all evaluations). Polymorphisms at codons 136, 154 and 171 were determined by denaturing gradient gel electrophoresis, followed by RFLP and sequencing. Scrapie, both clinical and subclinical, was associated with the genotypes ARQ/ARQ (88 of 110 sheep of that genotype), ARQ/TRQ (9 of 13), ARQ/AHQ (15 of 38) and VRQ/VRQ (9 of 17). Histopathological lesions were more severe in the clinical cases. Genotypes ARQ/ARR (26 sheep), ARQ/ARK (seven sheep), AHQ/ARR (one sheep), ARH/ARH (one sheep) and ARR/ARH (three sheep) were detected exclusively in healthy control sheep. In the purebred survey, four genotypes were present in the Chios sheep (ARQ/ARQ, ARQ/TRQ, ARQ/AHQ and ARQ/ARR) and four in the Karagouniko sheep (ARQ/ARQ, ARQ/AHQ, ARQ/ARR and ARQ/ARH).

scholarly journals Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

2016 ◽  
Author(s):  
Maureen M. Mundia ◽  
Alissa C. Magwood ◽  
Mark D. Baker
Keyword(s):  
Homologous Recombination ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Atp Hydrolysis ◽  
Dominant Negative ◽  
Strand Exchange ◽  
Wild Type ◽  
Hybridoma Cell Lines ◽  
Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

scholarly journals Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Merve Dede ◽  
Megan McLaughlin ◽  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Cell Lines ◽  
Protein Complexes ◽  
Gene Knockout ◽  
Essential Genes ◽  
Loss Of Function ◽  
Systematic Analysis ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions ◽  
Crispr Screen ◽  
A Cell

Abstract Background Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell. Results Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Conclusions Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.

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