Development of stable cell lines for production or regulated expression using matrix attachment regions

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

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Gastric Cancer Cell Lines Have DifferentMYC-Regulated Expression Patterns but Share a Common Core of Altered Genes

Canadian Journal of Gastroenterology and Hepatology ◽

10.1155/2018/5804376 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 2

Author(s):

Jersey Heitor da S. Maués ◽

Helem Ferreira Ribeiro ◽

Giovanny R. Pinto ◽

Luana de Oliveira Lopes ◽

Letícia M. Lamarão ◽

...

Keyword(s):

Gene Expression ◽

Gastric Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Common Core ◽

Gastric Cancer Cell ◽

Histological Subtypes ◽

Gene Sets ◽

Regulated Expression ◽

Gastric Cancer Cell Lines

MYCis an oncogene responsible for excessive cell growth in cancer, enabling transcriptional activation of genes involved in cell cycle regulation, metabolism, and apoptosis, and is usually overexpressed in gastric cancer (GC). By using siRNA and Next-Generation Sequencing (NGS), we identifiedMYC-regulated differentially expressed Genes (DEGs) in three Brazilian gastric cancer cell lines representing the histological subtypes of GC (diffuse, intestinal, and metastasis). The DEGs were picked usingSailfishsoftware, followed by Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis using KEGG. We found 11 significantly enriched gene sets by using enrichment score (ES), False Discovery Rate (FDR), and nominal P-values. We identified a total of 5.471 DEGs with correlation over (80%). In diffuse-type and in metastatic GC cell lines,MYC-silencing caused DEGs downregulation, while the intestinal-type GC cells presented overall DEGs upregulation afterMYCsiRNA depletion. We were able to detect 11 significant gene sets when comparing our samples to the hallmark collection of gene expression, enriched mostly for the following hallmarks: proliferation, pathway, signaling, metabolic, and DNA damage response. When we analyzed our DEGs considering KEGG metabolic pathways, we found 12 common branches covering a wide range of biological functions, and three of them were common to all three cell lines: ubiquitin-mediated proteolysis, ribosomes, and system and epithelial cell signaling inHelicobacter pyloriinfection. The GC cell lines used in this study share 14MYC-regulated genes, but their gene expression profile is different for each histological subtype of GC. Our results present a computational analysis ofMYC-related signatures in GC, and we present evidence that GC cell lines representing distinct histological subtypes of this disease have differentMYC-regulated expression profiles but share a common core of altered genes. This is an important step towards the understanding ofMYC’s role in gastric carcinogenesis and an indication of probable new drug targets in stomach cancer.

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Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells

Journal of Virology ◽

10.1128/jvi.79.22.13963-13973.2005 ◽

2005 ◽

Vol 79 (22) ◽

pp. 13963-13973 ◽

Cited By ~ 120

Author(s):

Zhaohui Cai ◽

Chen Zhang ◽

Kyung-Soo Chang ◽

Jieyun Jiang ◽

Byung-Chul Ahn ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Culture System ◽

Infectious Virus ◽

Hcv Infection ◽

Hcv Rna ◽

Human Hepatoma ◽

Stable Cell Lines ◽

Increased Risk

ABSTRACT Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5′ end and a hepatitis delta virus ribozyme at the 3′ end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 108 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

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