A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines

Abstract Background Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti. Methods An isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection. Results Genome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage. Conclusions Mosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies. Graphic abstract "Image missing"

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Growth of Nosema cuniculi in established cell lines

Laboratory Animals ◽

10.1258/002367775780994835 ◽

1975 ◽

Vol 9 (1) ◽

pp. 61-68 ◽

Cited By ~ 9

Author(s):

T. Waller

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Growth Patterns ◽

Rabbit Kidney ◽

Kidney Cells ◽

Scale Production ◽

Simple Method ◽

Established Cell Lines ◽

Established Cell ◽

Canine Kidney

Growth patterns of Nosema cuniculi ( Encephalitozoon cuniculi) in cell cultures of bovine kidney, canine kidney, feline lung, and rabbit kidney were studied. All cell cultures used were easy to manage and the last 3 are commercially-available established cell lines. The dog kidney cells were the most suitable for large-scale production of Nosema. When grown in plastic flasks with a bottom area of 75 cm2, the weekly yield from Nosema-infected canine kidney cells during the 10th to 17th week after inoculation was between 4·1 x 107 and 9·9 x 107 spores per flask. An equilibrium was obtained between the Nosema infection and the kidney cells during this time. A simple method for estimating the numbel of harvested spores is also described.

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

Wellcome Open Research ◽

10.12688/wellcomeopenres.15447.1 ◽

2019 ◽

Vol 4 ◽

pp. 150 ◽

Cited By ~ 3

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.

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P019 (0158) FUNCTIONAL ANALYSIS OF CD30 IN HODGKIN AND ANAPLASTIC LARGE CELL LYMPHOMA CELL LINES BY CRISPR/CAS9-MEDIATED GENE KNOCKOUT

HemaSphere ◽

10.1097/01.hs9.0000547868.14041.f9 ◽

2018 ◽

Vol 2 (S3) ◽

pp. 10

Keyword(s):

Functional Analysis ◽

Cell Lines ◽

Anaplastic Large Cell Lymphoma ◽

Cell Lymphoma ◽

Gene Knockout ◽

Large Cell ◽

Lymphoma Cell ◽

Large Cell Lymphoma

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Antigenic Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and Identification of Host Restriction Factors

mSphere ◽

10.1128/msphere.00095-20 ◽

2020 ◽

Vol 5 (3) ◽

Cited By ~ 4

Author(s):

Jessica J. Harrison ◽

Jody Hobson-Peters ◽

Agathe M. G. Colmant ◽

Joanna Koh ◽

Natalee D. Newton ◽

...

Keyword(s):

Monoclonal Antibodies ◽

West Nile Virus ◽

Cell Lines ◽

Yellow Fever Virus ◽

West Nile ◽

Host Restriction ◽

Antiviral Responses ◽

Vertebrate Cell ◽

Wide Range ◽

Chimeric Viruses

ABSTRACT We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs. We also constructed chimeric viruses between BinJV and the vertebrate-infecting West Nile virus (WNV) by swapping the structural genes (prM and E) to produce BinJ/WNVKUN-prME and WNVKUN/BinJV-prME. This allowed us to assess the role of different regions of the BinJV genome in vertebrate host restriction and revealed that while BinJV structural proteins facilitated entry to vertebrate cells, the process was inefficient. In contrast, the BinJV replicative components in wild-type BinJV and BinJ/WNVKUN-prME failed to initiate replication in a wide range of vertebrate cell lines at 37°C, including cells lacking components of the innate immune response. However, trace levels of replication of BinJ/WNVKUN-prME could be detected in some cultures of mouse embryo fibroblasts (MEFs) deficient in antiviral responses (IFNAR−/− MEFs or RNase L−/− MEFs) incubated at 34°C after inoculation. This suggests that BinJV replication in vertebrate cells is temperature sensitive and restricted at multiple stages of cellular infection, including inefficient cell entry and susceptibility to antiviral responses. IMPORTANCE The globally important flavivirus pathogens West Nile virus, Zika virus, dengue viruses, and yellow fever virus can infect mosquito vectors and be transmitted to humans and other vertebrate species in which they cause significant levels of disease and mortality. However, the subgroup of closely related flaviviruses, known as lineage II insect-specific flaviviruses (Lin II ISFs), only infect mosquitoes and cannot replicate in cells of vertebrate origin. Our data are the first to uncover the mechanisms that restrict the growth of Lin II ISFs in vertebrate cells and provides new insights into the evolution of these viruses and the mechanisms associated with host switching that may allow new mosquito-borne viral diseases to emerge. The new reagents generated in this study, including the first Lin II ISF-reactive monoclonal antibodies and Lin II ISF mutants and chimeric viruses, also provide new tools and approaches to enable further research advances in this field.

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

Genome Biology ◽

10.1186/s13059-020-02173-2 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 2

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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GREEN SYNTHESIS OF PYRAZOLO [3,4]-PYRIMIDINE-THIONES USING IONIC LIQUID 2-METHYL-IMIDAZOLIUM-OXALATE AS POTENT EHRLICH ASCITES CARCINOMA RECEPTOR ANTAGONISTS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i9.34661 ◽

2019 ◽

pp. 283-287

Author(s):

GANESH N YALLAPPA ◽

NAGARAJA D ◽

CHANDRASHEKHAR U

Keyword(s):

Ionic Liquid ◽

Anticancer Activity ◽

Cell Lines ◽

Conventional Method ◽

Ultrasonic Method ◽

Ehrlich Ascites Carcinoma ◽

Simple Method ◽

Heterocyclic Molecules ◽

Ehrlich Ascites ◽

Ic50 Values

Objective: Pyrazolopyrimidines are heterocyclic molecules containing nitrogen as the main composition, and hence, they exhibit pharmacological efficacy. They are analogs of purines so that possessing wide applications in the field of medicinal chemistry. The main objective of this study is to synthesize different derivatives of pyrazole-pyrimidine classes by adopting simple methodology as well as by employing green chemistry. The purpose of the synthesis of these molecules is to study the antitumor activity against Ehrlich ascites carcinoma (EAC) cell lines. Methods: After literature studies, it makes us to involve in the research of synthetic organic chemistry, especially to synthesize new compounds of pyrazolopyrimidines. We are reported solvent-free synthesis of pyrazolo [3,4-d]-pyrimidine-thiones through ethyl acetoacetate, hydrazine hydrate, thiourea, and different benzaldehydes. An ionic liquid 2-methyl-imidazolium-oxalate catalyzed the reactions under ultrasonication bath. Both conventional and ultrasonic methods were employed and comparison studies have been made. It was found that ultrasonic method completed the reaction quicker than the conventional method. All the synthesized compounds were confirmed their structures by 1HNMR, Fourier transform infrared, 13C-NMR, and elemental analysis spectra. The compounds were tested for in vitro anticancer activity against EAC cell lines. Most compounds revealed significant anticancer activity relative to doxorubicin as a positive control with inhibitory concentration (IC50) values. Results: Ultrasonication method is a simple method under which all the reactions were completed at faster time (<7 min) compared to the convention method. Among eight molecules, 8a and 8d completed the reactions at a faster rate. We reported IC50 values of all the molecules, in which 8e and 8g were exhibited excellent potency against EAC cell lines at different concentrations . Conclusions: Ultrasonication method is an excellent method for the organic synthesis. We are herein reported that under this method, all the reactions are completed within 7 min. Hence, it is superior method than the conventional method. All synthesized molecules have shown good inhibitor potency against EAC cell lines. Among them, two molecules 8e and 8g have shown excellent inhibitor potency.

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A Simple and Efficient Method to Generate Gene-Knockout and Transgenic Cell Lines

DNA and Cell Biology ◽

10.1089/dna.2020.6183 ◽

2020 ◽

Author(s):

Jieyu Liu ◽

Yan Ge ◽

Na Wang ◽

Jing Wen ◽

Wei Wang ◽

...

Keyword(s):

Cell Lines ◽

Efficient Method ◽

Gene Knockout ◽

Transgenic Cell

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CD40 is Positively Correlated with the Expression of Nucleophosmin in Cisplatin-Resistant Bladder Cancer

Journal of Oncology ◽

10.1155/2020/3676751 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Chenshuo Luo ◽

Ting Lei ◽

Man Zhao ◽

Qian Meng ◽

Man Zhang

Keyword(s):

Bladder Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Gene Knockout ◽

Cancer Cell Lines ◽

Fold Change ◽

Bladder Cancer Cell ◽

Drug Resistant ◽

Go Analysis ◽

Differential Proteins

Objective. To verify and evaluate the value of CD40 as a noninvasive biomarker of cisplatin-resistant bladder cancer, we studied the expression of CD40 and the correlation between nucleophosmin (NPM1) and CD40 in cisplatin-resistant bladder cancer. Methods. Three cisplatin-resistant bladder cancer cell lines (T24/0.8DDP, BIU87/0.3DDP, and PUMC-91/0.6DDP) were studied, and lentivirus was used to silence NPM1 expression. The expression of CD40 and NPM1 in three NPM1 silencing bladder cancer cell lines were detected by fluorescence microscopy and Western Blot. The effects and proteomic bioinformatics of NPM1 gene knockout on cisplatin-resistant bladder cancer cells were analyzed by liquid chromatography-mass spectrometry (LC-MS) and gene ontology analysis (GO analysis). Results. The NPM1 gene was successfully silenced in three drug-resistant bladder cancer cell lines by lentivirus infection. The knockdown efficiency was 70%. After NPM1 gene knockout, 492 differential proteins were detected by LC-MS, whose fold change was more than 1.5 p<0.05. A total of 57022 peptides, 54347 unique peptides, and 6686 protein groups were identified in all proteins of the tested cells (FDR < 0.01). Hierarchical clustering and principal component analysis showed that 264 functional proteins were downregulated and 228 functional proteins were upregulated in the gene silencing group compared with those of the negative controls. By GO analysis, the proteins affected by NPM1 cover a large number of proteins with biological functions, which may play an important role in the development of tumor in 492 differential proteins. The CD40 was the most significantly downregulated protein after NPM1 silencing, with a difference of 2.6-fold change in abundance. Conclusions. There is a positive correlation between CD40 protein and NPM1 protein in drug-resistant bladder cancer. Because NPM1 can reflect the characteristics of bladder cancer, CD40 may be a more sensitive marker for monitoring the prognosis of bladder cancer.

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