Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer

Background: Hundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. Results: Modulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId). Conclusions: In this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types

Targeted genomic integration of a selectable floxed dual fluorescence reporter in human embryonic stem cells.

The differentiation of pluripotent stem cells involves transition through a series of specific cell states. To understand these cell fate decisions, the field needs improved genetic tools for the labeling, lineage tracing and selection of specific cell types from heterogeneous differentiating populations, particularly in the human embryonic stem cell (hESC) system. We used zinc finger nuclease technology to stably insert a unique, selectable, floxed dual-fluorescence reporter transgene into the AAVS1 locus of RUES2 hESCs. This "stoplight" transgene, mTmG-2a-Puro, strongly expresses membrane-localized tdTomato red fluorescent protein until Cre-dependent recombination causes a switch to expression of membrane-localized enhanced green fluorescent protein (eGFP) and puromycin resistance. First, to validate this system in undifferentiated cells, we transduced transgenic hESCs with a lentiviral vector driving constitutive expression of Cre and observed the expected phenotypic switch. Next, to demonstrate its utility in lineage-specific selection, we transduced differentiated cultures with a lentiviral vector in which the striated muscle-specific CK7 promoter drives Cre expression. This yielded near-homogenous populations of eGFP(+) hESC-derived cardiomyocytes. The mTmg-2a-Puro hESC line described here represents a useful new tool for both in vitro fate mapping studies and the selection of useful differentiated cell types

Cre expression mediates a fluorescence switch in undifferentiated mTmG-2a-Puro floxed reporter hESCs.

<p>(<b>A</b>) Flow cytometry showing changing numbers of tdTomato⁺ and eGFP⁺ RUES2 mTmG-2a-Puro cells at various timepoints after transduction with an EF1α-Cre lentivirus. (<b>B</b>) Quantitation of the fluorescence switch shown in panel A (n = 3 biological replicates). (<b>C</b>) Scatter plot of undifferentiated mTmG-2a-Puro cells treated with EF1α-Cre lentivirus and then selected with puromycin for 48 hours. Note that the resultant cultures were >99% eGFP⁺ by flow cytometry (i). Comparison of untreated (red) and EF1α-Cre treated, puromycin selected (blue) undifferentiated mTmG-2a-Puro cells (ii). Quantitation of flow cytometry data from three independent experiments in which cells were transduced with EF1α-Cre and puromycin selected (iii). (<b>D</b>) Phase contrast and fluorescent photomicrographs of undifferentiated mTmG-2a-Puro cells (i), mTmG-2a-Puro cells treated with EF1α-Cre lentivirus (ii), and mTmG-2a-Puro cells treated with EF1α-Cre and selected with puromycin (iii). Scale bars are 50 µm. Error bars represent +/− one standard error of the mean. (<b>E</b>) mTmG-2a-Puro cells transduced with EF1α-Cre lentivirus were immunostained with an anti-Cre recombinase antibody. Note that Cre (magenta) co-localizes only with eGFP⁺ cell nuclei. Scale bar is 10 µm.</p

Human Embryonic Stem Cell-Derived Cardiomyocytes Migrate in Response to Gradients of Fibronectin and Wnt5a

Lincoln meteorological observations have been taken at a range of sites over the years. A NIWA report (Mullan, A.B; Stuart, S.J; Hadfield, M.G; Smith, M.J (2010). Report on the Review of NIWA's 'Seven-Station' Temperature Series. NIWA Information Series No. 78. pp.129-154) records a number of these along with the work undertaken to reconcile the data between different sites. It is not yet clear which site(s) these measurements were taken at as we have not yet identified a correspondence with NIWA's records.The datasets had been stored as .DAT files. The .DAT files have been uploaded as is, and also standardised and converted into .csv format.Headers: The original .DAT files were stored without headers. Most of these could be recovered for the .csv by running the data through an old program that had been used in conjunction with the data, but one column remains "unknown".Missing data: In the .DAT files, missing measurements are variously recorded, depending on context, as 0, -9, -99 or (in the case of Cloud cover) 9. In the .csv these values have been removed and left blank.Units are most likely:* solar radiation - probably MJ/m2 (megajoules per square metre)* temperatures - Celsius (in early years possibly converted from an original measurement in Fahrenheit)* rainfall - millimetres* cloud - oktas (eighths of the sky taken up by cloud)* wind run - kilometres* vapour pressure - probably Pa (pascals

Using CK-7 Cre in combination with mTmG-2a-Puro cells enables cardiomyocyte purification.

<p>(<b>A</b>) Flow cytometry for cardiac troponin T (cTnT) indicating the percentage of cardiomyocytes present after mTmG-2a-Puro hESCs were subjected to a directed cardiac differentiation protocol. (<b>B</b>) Mean percentage of cTnT⁺ cells resulting from three independent differentiation runs. (<b>C</b>) The percentage of cells that immunostained for eGFP, tdTomato, and/or the cardiomyocyte marker α-actinin was determined before and after puromycin selection (n = 230–600 cells per condition for three independent differentiation runs). (<b>D</b>) Magnification at 10x (i and ii) and 60x (iii and iv) showing fluorescent photomicrographs of cardiac differentiated mTmG-2a-Puro cells before (i and iii) and after (ii and iv) puromycin selection. Scale bars are 200 µm in (i and ii) and 50 µm in (iii and iv). Error bars represent one standard error of the mean.</p