A Bioinformatics Pipeline for the Identification of CHO Cell Differential Gene Expression from RNA-Seq Data

Monger, Craig; Motheramgari, Krishna; McSharry, John; Barron, Niall; Clarke, Colin

doi:10.1007/978-1-4939-6972-2_11

Cited by 3 publications

(2 citation statements)

References 16 publications

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“…The high-throughput RNA-Seq technique, based on next-generation sequencing technology, has emerged as a useful tool for transcriptome analysis and for exploring unknown genes. RNA-Seq provides a significantly more precise measurement of transcripts than other methods and has been successfully used for gene discovery in various species [14,15]. In order to determine if this new sequenced gene could encode a functionally active protein, we generated a stable cell line that expressed rPres.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of amphibian SLC26A5 using RNA-Seq

Wang

et al. 2021

BMC Genomics

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Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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Section: Introductionmentioning

confidence: 99%

Identification and characterization of amphibian SLC26A5 using RNA-Seq

Wang

et al. 2021

BMC Genomics

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“…The highthroughput RNA-Seq technique, based on next-generation sequencing technology, has emerged as a useful tool for transcriptome analysis and for exploring unknown genes. RNA-Seq provides a signi cantly more precise measurement of transcripts than other methods and has been successfully used for gene discovery in various species [14,15]. In order to determine if this new sequenced gene could encode a functionally active protein, we generated a stable cell line that expressed frog prestin.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of amphibian SLC26A5 using RNA-Seq

Wang

Hao

et al. 2020

Preprint

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Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Due to lacking of the 3D structure, most of the mechanism of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage. The knowledge of frog SLC26A5 is quite limited with only one species has been identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, the American bullfrog (Rana catesbeiana). In our study, the SLC26A5 gene of bullfrog has been mapped, sequenced and cloned successively using RNA-SEq. The comparative study revealed an alignment with nearly 40% identity among bullfrogs and mammalian species. The predicted 3D protein structure showed that the frog prestin possessed a transmembrane domain (TM) and a STAS domain similar to the mammalian prestin. The function of prestin crucially relies on its integration into the cell membrane. Such localization was observed when a prestin-EGFP fusion protein was expressed in HEK293T cells. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of frog’s inner ear and HEK293T cells transfected with this new coding gene. We observed that HEK293T cells expressing frog prestin showed electrophysiological features similar to that of hair cells from the amphibian’s inner ear. Conclusions We mapped and sequenced the SLC26A5 of the American bullfrog from its inner ear cDNA using RNA-SEq. The frog SLC26A5 cDNA was 2,292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. After isolating the prestin gene of the frog, we generated a stable cell line transfected with this new coding gene and found it possessing similar electrophysiological features as the hair cells from the frog’s auditory organ. Our experiment demonstrated that the new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line.

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Role of network-mediated stochasticity in mammalian drug resistance

et al. 2019

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A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to bet-hedging, drug tolerance, and cell-fate switching. The magnitude and timescales of stochastic fluctuations can depend on the gene regulatory network. Currently, it is unclear how gene expression noise of specific networks impacts the evolution of drug resistance in mammalian cells. Answering this question requires adjusting network noise independently from mean expression. Here, we develop positive and negative feedback-based synthetic gene circuits to decouple noise from the mean for Puromycin resistance gene expression in Chinese Hamster Ovary cells. In low Puromycin concentrations, the high-noise, positive-feedback network delays long-term adaptation, whereas it facilitates adaptation under high Puromycin concentration. Accordingly, the low-noise, negative-feedback circuit can maintain resistance by acquiring mutations while the positive-feedback circuit remains mutation-free and regains drug sensitivity. These findings may have profound implications for chemotherapeutic inefficiency and cancer relapse.

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A Bioinformatics Pipeline for the Identification of CHO Cell Differential Gene Expression from RNA-Seq Data

Cited by 3 publications

References 16 publications

Identification and characterization of amphibian SLC26A5 using RNA-Seq

Identification and characterization of amphibian SLC26A5 using RNA-Seq

Identification and characterization of amphibian SLC26A5 using RNA-Seq

Role of network-mediated stochasticity in mammalian drug resistance

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