A Chromatin Immunoprecipitation Approach Based on BAC Transgenesis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE10845 | GEO | 2008/03/15
SECONDARY ACCESSION(S): PRJNA107333
REPOSITORIES: GEO
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Project description:We have devised a technology that enables chromatin immunoprecipitation (ChIP) experiments in cultured human cells by circumventing both the laborious production of protein-specific antibodies and the optimization of crosslinking/immunoprecipitation conditions for each protein. Our approach utilizes bacterial artificial chromosomes (BACs) to physiologically express chromatinbinding proteins fused to epitope tags that are recognized by widely available and highly specific antibodies. We demonstrate the robustness of our methodology for mapping chromatin binding sites of transcription factors. Keywords: ChIP-Chip Analysis This series contains ChIP-on-Chip data sets for three transcription factors (FOXA1 (Forkhead box A1), XBP1-S (spliced X-box binding protein 1) and VDR (Vitamin D receptor)) and control samples (INPUT) for partial of the human genome including chromosomes 4, 15, 18, and 20 in MCF7 cells. We mapped the binding sites of these three transcription factors in the BAC transgenic MCF7 cells in which we tagged the transcription factors with a modified LAP (localization and affinity purification) tag. Goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP) was used to perform ChIP experiments in those transgenic lines. For comparison, we also mapped FOXA1 and XBP1-S binding sites with the available antibodies against the endogenous proteins. Goat anti-FoxA1 (Abcam: ab5089) and goat anti-XBP1 (Santa Cruz Biotech: sc-7160X) were used in these studies. Independent biological triplicates were performed for each transcription factor, as well as the control (Input).
2010-05-25 | E-GEOD-10845 | biostudies-arrayexpress
Project description:BACKGROUND: Bacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications. RESULTS: We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a approximately 70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations. CONCLUSION: The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.
| S-EPMC2768751 | biostudies-literature
Project description:Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.
| S-EPMC3479164 | biostudies-literature
Project description:In this study 405 patients with unexplained mental retardation and 89 unaffected parents were analyzed for DNA copy-number changes using a tiling-resolution genomewide microarray containing 32,447 BACs. This series contains 367 slides from 315 individuals previously not reported, plus an additional 239 slides from 179 individuals previously reported in the series GSE3191. Keywords: array CGH
2007-06-23 | GSE7391 | GEO
Project description:We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using UC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.
| S-EPMC2784134 | biostudies-literature
Project description:To gain insight into the specificity of target mRNAs, we enriched mRNAs bound to TRS by immunoprecipitation with anti-TRS antibody, then performed RNA immunoprecipitation and sequencing (RIP-seq) and compared the mRNAs with those enriched by immunoprecipitation with IgG, anti-AlaRS, anti-PRS and anti-IRS antibodies.
2019-02-05 | GSE120182 | GEO
Project description:Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.
| S-EPMC4825653 | biostudies-literature
Project description:The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate.
| S-EPMC5357965 | biostudies-literature
Project description:In frame of the IBSC sequenced BACs from Barley, that constitute the MTP of barley.
| PRJEB13020 | ENA
Project description:Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time and 4.5 hours machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the Plug and Play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naïve and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the 2-cell stage of embryonic development. PnP-ChIP-seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.
2021-02-10 | GSE120673 | GEO