Main Menu (Mobile)- Block

Main Menu - Block

custom | custom

Search Results

general_search_page-panel_pane_1 | views_panes

44 Janelia Publications

Showing 1-10 of 44 results
Your Criteria:
    09/21/17 | Genomic probes.
    Singer RH, Deng W, Lionnet T
    USPTO. 2017 Sep 21;A1:

    Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

    View Publication Page
    09/19/17 | Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments.
    Grimm JB, Brown TA, English BP, Lionnet T, Lavis LD
    Methods in Molecular Biology (Clifton, N.J.). 2017;1663:179-188. doi: 10.1007/978-1-4939-7265-4_15

    The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

    View Publication Page
    07/28/17 | Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.
    Kieffer-Kwon K, Nimura K, Rao SS, Xu J, Jung S, Pekowska A, Dose M, Stevens E, Mathe E, Dong P, Huang S, Ricci MA, Baranello L, Zheng Y, Ardori FT, Resch W, Stavreva D, Nelson S, McAndrew M, Casellas A, Finn E, Gregory C, St Hilaire BG, Johnson SM, Dubois W, Cosma MP, Batchelor E, Levens D, Phair RD, Misteli T, Tessarollo L, Hager G, Lakadamyali M, Liu Z, Floer M, Shroff H, Aiden EL, Casellas R
    Molecular Cell. 2017 Jul 28;67(4):566-78. doi: 10.1016/j.molcel.2017.07.013

    50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

    View Publication Page
    07/01/17 | mRNA quantification using single-molecule FISH in Drosophila embryos.
    Trcek T, Lionnet T, Shroff H, Lehmann R
    Nature Protocols. 2017 Jul;12(7):1326-1348. doi: 10.1038/nprot.2017.030

    Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.

    View Publication Page
    06/05/17 | Quantitative mRNA imaging throughout the entire Drosophila brain.
    Long X, Colonell J, Wong AM, Singer RH, Lionnet T
    Nature Methods. 2017 Jun 05;14(7):703-6. doi: 10.1038/nmeth.4309

    We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).

    View Publication Page
    02/27/17 | A variant Sp1 (R218Q) transcription factor might enhance HbF expression in β(0) -thalassaemia homozygotes.
    Jiang Z, Luo H, Farrell JJ, Zhang Z, Schulz VP, Albarawi D, Steinberg MH, Al-Allawi NA, Gallagher PG, Forget BG, Chui DH
    British Journal of Haematology. 2017 Feb 27;180(5):755-7. doi: 10.1111/bjh.14445
    09/15/16 | Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
    Zhang Z, English BP, Grimm JB, Kazane SA, Hu W, Tsai A, Inouye C, You C, Piehler J, Schultz PG, Lavis LD, Revyakin A, Tjian R
    Genes and Development. 2016 Sep 15;30:2106-18. doi: 10.1101/gad.285395.116

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

    View Publication Page
    07/29/16 | Bright photoactivatable fluorophores for single-molecule imaging.
    Lavis LD, Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T
    bioRxiv. 2016 Jul 29:066779. doi: 10.1101/066779

    Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

    View Publication Page
    07/12/16 | Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers.
    Breda L, Motta I, Lourenco S, Gemmo C, Deng W, Rupon JW, Abdulmalik OY, Manwani D, Blobel GA, Rivella S
    Blood. 2016 Jul 12:. doi: 10.1182/blood-2016-01-691089

    Overcoming the silencing of the fetal γ-globin gene has been a long standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called LCR, dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells the LCR can be re-directed from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom designed zinc finger proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacological reactivation of fetal hemoglobin (HbF), hematopoietic cells from SCD patients were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double of that observed with decitabine and pomalidomide; butyrate had an intermediate effect while tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle Hb (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared to the drug regimens support its therapeutic potential for the treatment of SCD.

    View Publication Page
    05/05/16 | Real-time quantification of single RNA translation dynamics in living cells.
    Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis LD, Grimm JB, Viswanathan S, Looger LL
    Science. 2016 May 05;352(6292):1425-9. doi: 10.1126/science.aaf0899

    Although mRNA translation is a fundamental biological process, it has never been imaged in real-time with single molecule precision in vivo. To achieve this, we developed Nascent Chain Tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify single mRNA protein synthesis dynamics. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 s. Polysomes contain ~1 ribosome every 200-900 nucleotides and are globular rather than elongated in shape. By developing multi-color probes, we show most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

    View Publication Page