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  • 1
    Language: English
    In: Developmental biology, 15 April 2013, Vol.376(2), pp.224-35
    Description: Long non-coding RNAs (lncRNAs) have been recently recognized as a major class of regulators in mammalian systems. LncRNAs function by diverse and heterogeneous mechanisms in gene regulation, and are key contributors to development, neurological disorders, and cancer. This emerging importance of lncRNAs, along with recent reports of a functional lncRNA encoded by the mouse Dlx5-Dlx6 locus, led us to interrogate the biological significance of another distal-less antisense lncRNA, the previously uncharacterized Dlx1 antisense (Dlx1as) transcript. We have functionally ablated this antisense RNA via a highly customized gene targeting approach in vivo. Mice devoid of Dlx1as RNA are viable and fertile, and display a mild skeletal and neurological phenotype reminiscent of a Dlx1 gain-of function phenotype, suggesting a role for this non-coding antisense RNA in modulating Dlx1 transcript levels and stability. The reciprocal relationship between Dlx1as and Dlx1 places this sense-antisense pair into a growing class of mammalian lncRNA-mRNA pairs characterized by inverse regulation.
    Keywords: Gene Expression Regulation, Developmental ; Homeodomain Proteins -- Genetics ; RNA, Antisense -- Genetics ; Transcription Factors -- Genetics
    ISSN: 00121606
    E-ISSN: 1095-564X
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  • 2
    Language: English
    In: PLoS ONE, 2011, Vol.6(12), p.e28885
    Description: Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo , but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo , the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 ( Sox9 ) and enhanced green fluorescent protein ( EGFP ) linked by the IRES ( Sox9 IRES-EGFP ) or the F2A ( Sox9 F2A-EGFP ) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG 3 -Bapx1 F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog ( Bapx1 ), Cre recombinase and EGFP ( Bapx1 F2A-Cre-F2A-EGFP ). While the ‘skipping’ was highly efficient between Bapx1 and Cre , the ‘skipping’ between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the ‘skipping’ at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Developmental Biology
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: BMC Genetics, July 16, 2010, Vol.11, p.68
    Description: The inner ear is one of the most complex and detailed organs in the vertebrate body and provides us with the priceless ability to hear and perceive linear and angular acceleration (hence maintain balance). The development and morphogenesis of the inner ear from an ectodermal thickening into distinct auditory and vestibular components depends upon precise temporally and spatially coordinated gene expression patterns and well orchestrated signaling cascades within the otic vesicle and upon cellular movements and interactions with surrounding tissues. Gene loss of function analysis in mice has identified homeobox genes along with other transcription and secreted factors as crucial regulators of inner ear morphogenesis and development. While otic induction seems dependent upon fibroblast growth factors, morphogenesis of the otic vesicle into the distinct vestibular and auditory components appears to be clearly dependent upon the activities of a number of homeobox transcription factors. The Pax2 paired-homeobox gene is crucial for the specification of the ventral otic vesicle derived auditory structures and the Dlx5 and Dlx6 homeobox genes play a major role in specification of the dorsally derived vestibular structures. Some Micro RNAs have also been recently identified which play a crucial role in the inner ear formation.
    Keywords: Transcription Factors -- Research ; Transcription Factors -- Physiological Aspects ; Labyrinth (Ear) -- Physiological Aspects ; Labyrinth (Ear) -- Genetic Aspects ; Labyrinth (Ear) -- Research ; Microrna -- Physiological Aspects ; Microrna -- Research ; Gene Expression -- Research
    ISSN: 1471-2156
    Source: Cengage Learning, Inc.
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  • 4
    In: Nature, 2010, Vol.468(7321), p.316
    Description: The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells. [PUBLICATION ]
    Keywords: Animals–Genetics ; Base Sequence–Metabolism ; Cell Line–Cytology ; DNA-Binding Proteins–Metabolism ; DNA-Binding Proteins–Genetics ; Embryonic Stem Cells–Cytology ; Embryonic Stem Cells–Metabolism ; Enhancer Elements, Genetic–Genetics ; Fibroblasts–Genetics ; Fibroblasts–Cytology ; Gene Expression Regulation–Metabolism ; Genome, Human–Genetics ; Humans–Genetics ; Induced Pluripotent Stem Cells–Metabolism ; Induced Pluripotent Stem Cells–Genetics ; Mice–Metabolism ; Nuclear Reprogramming–Metabolism ; Octamer Transcription Factor-3–Metabolism ; Octamer Transcription Factor-3–Metabolism ; RNA Interference–Metabolism ; Repressor Proteins–Metabolism ; Repressor Proteins–Metabolism ; Soxb1 Transcription Factors–Metabolism ; Stem Cells ; Candidates ; Gene Expression ; Proteins ; DNA-Binding Proteins ; Nfrkb Protein, Human ; Octamer Transcription Factor-3 ; Pou5f1 Protein, Human ; Prdm14 Protein, Human ; Repressor Proteins ; Sox2 Protein, Human ; Soxb1 Transcription Factors;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 5
    In: Nature Biotechnology, 2013, Vol.31(7), p.615
    Description: Despite their apparent diversity, many problems in the analysis of high-throughput sequencing data are merely special cases of two general problems, signal detection and signal estimation. Here we adapt formally optimal solutions from signal processing theory to analyze signals of DNA sequence reads mapped to a genome. We describe DFilter, a detection algorithm that identifies regulatory features in ChIP-seq, DNase-seq and FAIRE-seq data more accurately than assay-specific algorithms. We also describe EFilter, an estimation algorithm that accurately predicts mRNA levels from as few as 1-2 histone profiles (R 0.9). Notably, the presence of regulatory motifs in promoters correlates more with histone modifications than with mRNA levels, suggesting that histone profiles are more predictive of cis-regulatory mechanisms. We show by applying DFilter and EFilter to embryonic forebrain ChIP-seq data that regulatory protein identification and functional annotation are feasible despite tissue heterogeneity. The mathematical formalism underlying our tools facilitates integrative analysis of data from virtually any sequencing-based functional profile. [PUBLICATION ]
    Keywords: Algorithms–Methods ; Base Sequence–Methods ; Genome–Methods ; High-Throughput Nucleotide Sequencing–Methods ; Sequence Analysis, DNA–Methods ; Signal-To-Noise Ratio–Methods ; Signal Processing ; Optimization Algorithms ; Genomics;
    ISSN: 1087-0156
    E-ISSN: 15461696
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  • 6
    Language: English
    In: Cytotechnology, 2018, Vol.70(1), pp.185-192
    Description: Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q 2 PISH allows for the analysis of both qualitative and quantitative (q 2 ) gene expression on cultured cells for quality control measures with single cell resolution. q 2 PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1 , Col2a1 , Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q 2 PISH serves as an accurate quality control with single cell resolution for cultured cells.
    Keywords: Alkaline phosphatase ; Bovine ; NBT/BCIP ; PISH ; pNPP ; RNA in situ hybridization ; Single cell gene expression
    ISSN: 0920-9069
    E-ISSN: 1573-0778
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  • 7
    Language: English
    In: Acta Histochemica, March 2017, Vol.119(2), pp.150-160
    Description: Degeneration of the intervertebral disc (IVD) is a meritorious target for therapeutic cell based regenerative medicine approaches, however, controversy over what defines the precise identity of mature IVD cells and lack of single cell based quality control measures is of concern. Bos taurus and human IVDs are histologically more similar than is Mus musculus. The mature bovine IVD is well suited as model system for technology development to be translated into therapeutic cell based regenerative medicine applications. We present a reproducible non-enzymatic protocol to isolate cell progenitor populations of three distinct areas of the mature bovine IVD. Bovine specific RNA probes were validated in situ and employed to assess fate changes, heterogeneity, stem cell characteristics and differentiation potential of the cultures. Quality control measures with single cell resolution like RNA in situ hybridization to assess culture heterogeneity (PISH) followed by optimization of culture conditions could be translated to human IVD cell culture to increase the safety of cell based regenerative medicine.
    Keywords: RNA in Situ Hybridization ; Regenerative Medicine ; Single Cell Quality Control ; Annulus Fibrosus ; Nucleus Pulposus
    ISSN: 0065-1281
    E-ISSN: 16180372
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  • 8
    Language: English
    In: Genomics Data, 01 December 2016, Vol.10(C), pp.83-84
    Description: The adult bovine (Bos taurus) intervertebral disc is primarily comprised of two major tissue types: The outer annulus fibrosus (AF) and the central nucleus pulposus (NP). We isolated several primary cell lineages of passage (P) 0 cells from the AF tissue omitting typically used enzymatic tissue digestion protocols. The cells grow past p10 without signs of senescence in DMEM + 10% FCS on 0.1% gelatin coated/uncoated surfaces of standard cell culture plates and survive freeze-thawing. Preliminary analysis of the AF derived cells for expression of the two structural genes Col1a1 and Col2a1 was performed by PISH recapitulating the expression observed in vivo.
    Keywords: Bovine ; Intervertebral Disc ; Annulus Fibrosus ; Cell Line ; In Situ Hybridization ; Biology
    ISSN: 2213-5960
    E-ISSN: 2213-5960
    Source: Directory of Open Access Journals (DOAJ)
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  • 9
    Language: English
    In: Genomics data, December 2016, Vol.10, pp.83-84
    Description: The adult bovine ( intervertebral disc is primarily comprised of two major tissue types: The outer annulus fibrosus (AF) and the central nucleus pulposus (NP). We isolated several primary cell lineages of passage (P) 0 cells from the AF tissue omitting typically used enzymatic tissue digestion protocols. The cells grow past p10 without signs of senescence in DMEM + 10% FCS on 0.1% gelatin coated/uncoated surfaces of standard cell culture plates and survive freeze-thawing. Preliminary analysis of the AF derived cells for expression of the two structural genes and was performed by PISH recapitulating the expression observed .
    Keywords: Annulus Fibrosus ; Bovine ; Cell Line ; In Situ Hybridization ; Intervertebral Disc
    ISSN: 2213-5960
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 10
    Language: English
    In: BMC Genetics, 01 July 2010, Vol.11(1), p.68
    Description: Abstract The inner ear is one of the most complex and detailed organs in the vertebrate body and provides us with the priceless ability to hear and perceive linear and angular acceleration (hence maintain balance). The development and morphogenesis of the inner ear from an ectodermal thickening into distinct auditory and vestibular components depends upon precise temporally and spatially coordinated gene expression patterns and well orchestrated signaling cascades within the otic vesicle and upon cellular movements and interactions with surrounding tissues. Gene loss of function analysis in mice has identified homeobox genes along with other transcription and secreted factors as crucial regulators of inner ear morphogenesis and development. While otic induction seems dependent upon fibroblast growth factors, morphogenesis of the otic vesicle into the distinct vestibular and auditory components appears to be clearly dependent upon the activities of a number of homeobox transcription factors. The Pax2 paired-homeobox gene is crucial for the specification of the ventral otic vesicle derived auditory structures and the Dlx5 and Dlx6 homeobox genes play a major role in specification of the dorsally derived vestibular structures. Some Micro RNAs have also been recently identified which play a crucial role in the inner ear formation.
    Keywords: Biology
    ISSN: 1471-2156
    E-ISSN: 1471-2156
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