Profiling conserved microRNA expression in recombinant CHO cell lines using Illumina sequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Hammond S
PROVIDER: S-EPMC3330154 | biostudies-literature | 2012 Jun
REPOSITORIES: biostudies-literature
Publications
Profiling conserved microRNA expression in recombinant CHO cell lines using Illumina sequencing.
Hammond Stephanie S Swanberg Jeffrey C JC Polson Shawn W SW Lee Kelvin H KH
Biotechnology and bioengineering 20120123 6
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate multiple aspects of cell physiology. The differential expression of conserved miRNAs in two Chinese hamster ovary (CHO) cell lines producing recombinant proteins was examined relative to the CHO-K1 cell line. A total of 190 conserved CHO miRNAs were identified through homology with known human and rodent miRNAs. More than 80% of these miRNAs showed differential expression in recombinant CHO cell lines. The small RNA sequencing data were ...[more]
Similar Datasets
Project description:The aim of this work was to establish the microRNA profile of SNK6 and SNT16, two Epstein-Barr virus (EBV)-infected cell lines derived from nasal NK/T-cell lymphoma (NKTL). The oncogenic EBV is strongly associated with the pathogenesis of nasal and extranodal NK/T-cell lymphoma and expresses 44 mature microRNAs and two noncoding EBV-encoded RNAs (EBERs). miRNAs are 19-25nt noncoding RNAs that affect host and viral gene expression post-transcriptionally. Deregulated miRNA patterns are frequently linked to a variety of human cancers including lymphomas. miRNA profiling of the two NK/T cell lines vs. primary cells revealed 10 and 4 up-regulated and 10 and 12 down-regulated miRNAs in SNK6 and SNT16 cells respectively. The results were validated by qRT-PCR for selected miRNAs. Target gene analyses confirmed cullin 5 (CUL5) and sphingosin-1-phosphate receptor 1 (S1PR1) as targets for the down-regulated hsa-miR-148a and viral ebv-miR-BART16 respectively. As recently demonstrated for the regulation of IL1-alpha by miR-142-3p, coexpression of the EBERs selectively exerted corepression of S1PR1 by BART16 but not of CUL5 by miR-148a, indicating selective corepression by the EBERs.
| S-EPMC4821355 | biostudies-literature
Project description:microRNA (miRNA) expression profiles based on the highly powerful Illumina sequencing technology rely on the construction of cDNA libraries in which adaptor ligation is known to deeply favor some miRNAs over others. This introduces erroneous measurements of the miRNA abundances and relative miRNA quantities in biological samples. Here, by using the commercial miRXplore Universal Reference that contains an equimolar mixture of 963 animal miRNAs and TruSeq or bulged adaptors, we describe a method for correcting ligation biases in expression profiles obtained with standard protocols of cDNA library construction and provide data for quantifying the true miRNA abundances in biological samples. Ligation biases were evaluated at three ratios of miRNA to 3'-adaptor and four numbers of polymerase chain reaction amplification cycles by calculating efficiency captures/correcting factors for each miRNA. We show that ligation biases lead to over- or under-expression covering a 105 amplitude range. We also show that, at each miRNA:3'-adaptor ratio, coefficients of variation (CVs) of efficiency captures calculated over the four number of amplification cycles using sliding windows of 10 values ranged from 0.1 for the miRNAs of high expression to 0.6 for the miRNAs of low expression. Efficiency captures of miRNAs of high and low expression in profiles are therefore differently impacted by the number of amplification cycles. Importantly, we observed that at a given number of amplification cycles, CVs of efficiency captures calculated over the three miRNA:3'-adaptor ratios displayed a steady value of 0.3 +/- 0.05 STD for miRNAs of high and low expression. This allows, at a given number of amplification cycles, accurate comparison of miRNA expression between biological samples over a substantial expression range. Finally we provide tables of correcting factors that allow to measure the abundances of 963 miRNAs in biological samples from TruSeq-based expression profiles and, an example of their use by characterizing miRNAs of the let-7, miR-26, miR-29, and miR-30 families as the more abundant miRNAs of the rat adult cerebellum.
| S-EPMC6491513 | biostudies-literature
Project description:This study aimed to evaluate the difference in gut microbiomes between preterm and term infants using third-generation long-read sequencing (Oxford Nanopore Technologies, ONT) compared with an established gold standard, Illumina (second-generation short-read sequencing). A total of 69 fecal samples from 51 term (T) and preterm (P) infants were collected at 7 and 28 days of life. Gut colonization profiling was performed by 16S rRNA gene sequencing using ONT. We used Illumina to validate and compare the patterns in 13 neonates. Using bioinformatic analysis, we identified features that differed between P and T. Both T1 and P1 microbiomes were dominated by Firmicutes (Staphylococcus and Enterococcus), whereas sequentially showed dominant transitions to Lactobacillus (p < 0.001) and Streptococcus in T2 (p = 0.001), and pathogenic bacteria (Klebsiella) in P2 (p = 0.001). The abundance of beneficial bacteria (Bifidobacterium and Lactobacillus) increased in T2 (p = 0.026 and p < 0.001, respectively). These assignments were correlated with the abundance at the species-level. Bacterial α-diversity increased in T (p = 0.005) but not in P (p = 0.156), and P2 showed distinct β-diversity clustering than T2 (p = 0.001). The ONT reliably identified pathogenic bacteria at the genus level, and taxonomic profiles were comparable to those identified by Illumina at the genus level. This study shows that ONT and Illumina are highly correlated. P and T had different microbiome profiles, and the α- and β-diversity varied. ONT sequencing has potential for pathogen detection in neonates in clinical settings.
| S-EPMC10225602 | biostudies-literature
Project description:MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. These applications, however, require a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of ~400 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, 415 previously reported CHO miRNAs were mapped to the reference genomes, and subsequently assigned to a distinct genomic miRNA locus. Sequences of the respective precursor-miRNAs were extracted from both reference genomes, folded in silico to verify correct structures and cross-compared. In the end, 212 genomic loci and pre-miRNA sequences representing 319 expressed mature miRNAs (approximately 50% of miRNAs represented matching pairs of 5' and 3' miRNAs) were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four likely polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, these data on the genomic context of miRNA expression in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.
| S-EPMC3314935 | biostudies-literature
Project description:Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise minimization may therefore be a novel strategy to reduce apparent expression instability and simplify cell line selection.
| S-EPMC2793030 | biostudies-literature
Project description:MicroRNAs (miRNAs/miRs) are sensitive biomarkers and endogenous repressors of gene expression by decreasing mRNA stability and interfering with mRNA translation. Despite a number of investigations revealing the dysregulation of miRNA expression associated with cardiotoxicity induced by doxorubicin (Dox), perturbation of miRNAs directly resulting from Dox at early stage in cardiomyocytes and the target gene interaction remain largely unknown. In the present study, high-throughput deep-sequencing was used to analyze changes in global miRNA expression in H9c2 cardiomyocytes exposed to 5 µg/ml Dox for 0, 12 or 24 h. Compared with the 0-h time point, the expression levels of 386 unique miRNAs were altered. Based on miRNA expression and fold-change, the target genes of 76 selected miRNAs were further analyzed using gene interaction networks and pathway enrichment analysis. These miRNAs were involved in the regulation of different pathways, whose functions included apoptosis, cell proliferation, extracellular matrix remodeling, oxidative stress and lipid metabolism. These differentially expressed miRNAs included let-7 family, miR-29b-3p, miR-378-3/5p, miR-351-3p, miR-664-3p, miR-455-3p, miR-298-3p, miR-702-5p, miR-128-1-5p, miR-671 and miR-421-5p. The present data indicated that global wide miRNA profiling in Dox-induced cardiomyocytes may provide a novel mechanistic insight into understanding Dox-induced heart failure and cardiotoxicity, as well as novel biomarkers and therapeutic targets.
| S-EPMC8170198 | biostudies-literature
Project description:The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.
| S-EPMC2896516 | biostudies-literature
Project description:The testis is a highly specialized tissue that plays dual roles in ensuring fertility by producing spermatozoa and hormones. Spermatogenesis is a complex process, resulting in the production of mature sperm from primordial germ cells. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. The gene expression pattern of testis in Chinese mitten crab (Eriocheir sinensis) has not been extensively studied, and limited genetic research has been performed on this species. The advent of high-throughput sequencing technologies enables the generation of genomic resources within a short period of time and at minimal cost. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for testis of E. sinensis. In two runs, we produced 25,698,778 sequencing reads corresponding with 2.31 Gb total nucleotides. These reads were assembled into 342,753 contigs or 141,861 scaffold sequences, which identified 96,311 unigenes. Based on similarity searches with known proteins, 39,995 unigenes were annotated based on having a Blast hit in the non-redundant database or ESTscan results with a cut-off E-value above 10(-5). This is the first report of a mitten crab transcriptome using high-throughput sequencing technology, and all these testes transcripts can help us understand the molecular mechanisms involved in spermatogenesis and testis maturation.
| S-EPMC3307765 | biostudies-literature
Project description:Background:Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results:In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15?g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95-100% of productivity after 40?days of continuous culture (~ 48-56 generations). Conclusions:Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.How to cite: Li J, Wei S, Cao C, et al. Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.002.
| S-EPMC7125944 | biostudies-literature
Project description:BackgroundMicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion.MethodsMicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR.ResultsEach cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50-60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes.ConclusionsThese data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.
| S-EPMC9307586 | biostudies-literature