scholarly journals High-throughput Sequencing for Species Authentication and Contamination Detection of 63 Cell Lines

2021 ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

Abstract Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

scholarly journals High-throughput sequencing for species authentication and contamination detection of 63 cell lines

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

AbstractCell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

scholarly journals Incidences of problematic cell lines are lower in papers that use RRIDs to identify cell lines

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Zeljana Babic ◽  
Amanda Capes-Davis ◽  
Maryann E Martone ◽  
Amos Bairoch ◽  
I Burak Ozyurt ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Biomedical Research ◽  
Cell Line Authentication ◽  
Pubmed Central ◽  
International Cell ◽  
Unique Cell

The use of misidentified and contaminated cell lines continues to be a problem in biomedical research. Research Resource Identifiers (RRIDs) should reduce the prevalence of misidentified and contaminated cell lines in the literature by alerting researchers to cell lines that are on the list of problematic cell lines, which is maintained by the International Cell Line Authentication Committee (ICLAC) and the Cellosaurus database. To test this assertion, we text-mined the methods sections of about two million papers in PubMed Central, identifying 305,161 unique cell-line names in 150,459 articles. We estimate that 8.6% of these cell lines were on the list of problematic cell lines, whereas only 3.3% of the cell lines in the 634 papers that included RRIDs were on the problematic list. This suggests that the use of RRIDs is associated with a lower reported use of problematic cell lines.

High-throughput sequencing identifies HIV-1-replication- and latency-related miRNAs in CD4+ T cell lines

2017 ◽  
Vol 162 (7) ◽  
pp. 1933-1942 ◽  
Author(s):  
Xiangyun Lu ◽  
Jin Yang ◽  
Haibo Wu ◽  
Zongxing Yang ◽  
Changzhong Jin ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cd4 T Cell ◽  
T Cell Lines ◽  
Hiv 1

scholarly journals An Isogenic Cell Line Panel for Sequence-based Screening of Targeted Anti-cancer Drugs

2021 ◽  
Author(s):  
Ashley L Cook ◽  
Nicolas Wyhs ◽  
Surojit B Sur ◽  
Blair Ptak ◽  
Maria Popoli ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Growth Inhibitor ◽  
Suppressor Gene ◽  
Loss Of Function ◽  
Cancer Pathways ◽  
Isogenic Cell Line ◽  
Cell Line Panel

We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-193a as a Regulator of Jagged1 In Marrow Stromal Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3847-3847
Author(s):  
Ilango Balakrishnan ◽  
Xiaodong Yang ◽  
Beverly Torok-Storb ◽  
Jay Hesselberth ◽  
Manoj Pillai
Keyword(s):  
Binding Site ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Direct Interaction ◽  
Over Expression ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Immune Precipitation ◽  
Mrna Binding

Abstract Abstract 3847 MicroRNAs (miRNAs) are small non-coding RNAs with important roles in the post-transcriptional regulation of up to 30% of all vertebrate genes. Traditional methods to determine miRNA-mRNA interactions have included transcriptional profiling of miRNAs, bio-informatic prediction of miRNA-mRNA binding, analysis of 3` untranslated region (3`UTR) binding of miRNAs and over-expression of miRNAs in relevant cell types. These studies however fall short of demonstrating direct interaction between a miRNA and its target mRNAs. We applied a recently described biochemical technique of high throughput sequencing following cross-linked immune precipitation (HITS-CLIP) to dissecting the miRNA-mRNA interactions in two functionally distinct human marrow stromal cell lines. HITS-CLIP relies on the ability of ultraviolet (UV) radiation to cross-link RNA to proteins they are bound to, followed by immune-precipitation of the RNA-protein complex to isolate the cross-linked RNA and sequencing by high throughput techniques. As miRNA-mRNA interactions occur in close proximity to the argonaute proteins (AGO), an anti-argonaute monoclonal antibody was used to isolate the Ago-miRNA-mRNA complexes. The two stromal cell lines analyzed by HITS-CLIP (designated HS5 and HS27a) were isolated from a normal marrow primary long term culture (LTC), immortalized and extensively characterized for both function and expression profiles (mRNA and miRNA). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (CXCL12, Angiopoietin-1, Jag1 etc). In keeping with this, HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. Sequence reads from the HITS-CLIP analysis from each of the cell lines were aligned to the human genome using the UCSC genome browser to identify Ago-mRNA and Ago-miRNA binding sites in both the cell lines. Interestingly, corresponding datasets from HS5 and HS27a were similar for the majority of mRNAs and miRNAs, but distinct for those mRNAs (such as Jag1, CXCL12, IL6 and GCSF) and miRNAs (such as miR-886-3p, miR-221, miR-181a and miR-193a) known to be differentially expressed between the two cell lines. We then validated the use of the HITS-CLIP strategy in stromal cells by analyzing one such Ago-mRNA binding site for Jagged1 (Jag1). Jag1 is a ligand for Notch1 and is expressed in those cells that support the hematopoietic stem cell (HSC) niche. The Notch pathway is a highly conserved signaling system critical in regulating several tissue systems including hematopoietic cells. This binding site, 1749 bp downstream of the transcriptional start-site for Jag1 was significantly more enriched in HS5 compared to HS27a. The site was also a predicted binding site for miR-193a, a miRNA over-expressed in HS5 compared to HS27a cells. Over-expression of miR-193a in HS27a cells resulted in the down-regulation of Jag1 protein (as measured by Western blotting). To confirm the direct interaction between Jag1 and miR-193a, we cloned this purported binding site downstream of the luciferase gene and co-transfected the plasmid with miR-193a. Luciferase activity was down-regulated greater than 50% when compared to control transfections suggesting a direct effect of miR-193a on Jag1 transcript. In summary, our data suggest that HITS-CLIP methodology can be used to define in vivo spatial interactions between miRNA and mRNAs in the marrow microenvironment (ME). It can also be used to define miRNA-based regulation of specific genes such as Jag1, which are critical to defining functional niches in the ME. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-Throughput RNA Sequencing-Based Virome Analysis of 50 Lymphoma Cell Lines from the Cancer Cell Line Encyclopedia Project

2014 ◽  
Vol 89 (1) ◽  
pp. 713-729 ◽  
Author(s):  
Subing Cao ◽  
Michael J. Strong ◽  
Xia Wang ◽  
Walter N. Moss ◽  
Monica Concha ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cancer Cell Line ◽  
Lymphoma Cell ◽  
Viral Gene ◽  
Barr Virus ◽  
Murine Leukemia

ABSTRACTUsing high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.IMPORTANCEViruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

scholarly journals Scrutinizing Virus Genome Termini by High-Throughput Sequencing

PLoS ONE ◽  
2014 ◽  
Vol 9 (1) ◽  
pp. e85806 ◽  
Author(s):  
Shasha Li ◽  
Hang Fan ◽  
Xiaoping An ◽  
Huahao Fan ◽  
Huanhuan Jiang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Genome ◽  
Genome Termini
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