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A modular open platform for systematic functional studies under physiological conditions.

ABSTRACT: Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.

SUBMITTER: Mulholland CB

PROVIDER: S-EPMC4787826 | biostudies-other | 2015 Sep

REPOSITORIES: biostudies-other

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A modular open platform for systematic functional studies under physiological conditions.

Mulholland Christopher B CB Smets Martha M Schmidtmann Elisabeth E Leidescher Susanne S Markaki Yolanda Y Hofweber Mario M Qin Weihua W Manzo Massimiliano M Kremmer Elisabeth E Thanisch Katharina K Bauer Christina C Rombaut Pascaline P Herzog Franz F Leonhardt Heinrich H Bultmann Sebastian S

Nucleic acids research 20150524 17

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, ...[more]

PMID: 26007658

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Project description:Piezocrystals, especially the relaxor-based ferroelectric crystals, have been subject to intense investigation and development within the past three decades, motivated by the performance advantages offered by their ultrahigh piezoelectric coefficients and higher electromechanical coupling coefficients than piezoceramics. Structural anisotropy of piezocrystals also provides opportunities for devices to operate in novel vibration modes, such as the d36 face shear mode, with domain engineering and special crystal cuts. These piezocrystal characteristics contribute to their potential usage in a wide range of low- and high-power ultrasound applications. In such applications, conventional piezoelectric materials are presently subject to varying mechanical stress/pressure, temperature and electric field conditions. However, as observed previously, piezocrystal properties are significantly affected by a single such condition or a combination of conditions. Laboratory characterisation of the piezocrystal properties under these conditions is therefore essential to fully understand these materials and to allow electroacoustic transducer design in realistic scenarios. This will help to establish the extent to which these high performance piezocrystals can replace conventional piezoceramics in demanding applications. However, such characterisation requires specific experimental arrangements, examples of which are reported here, along with relevant results. The measurements include high frequency-resolution impedance spectroscopy with the piezocrystal material under mechanical stress 0-60 MPa, temperature 20-200 °C, high electric AC drive and DC bias. A laser Doppler vibrometer and infrared thermal camera are also integrated into the measurement system for vibration mode shape scanning and thermal conditioning with high AC drive. Three generations of piezocrystal have been tested: (I) binary, PMN-PT; (II) ternary, PIN-PMN-PT; and (III) doped ternary, Mn:PIN-PMN-PT. Utilising resonant mode analysis, variations in elastic, dielectric and piezoelectric constants and coupling coefficients have been analysed, and tests with thermal conditioning have been carried out to assess the stability of the piezocrystals under high power conditions.

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A modular open platform for systematic functional studies under physiological conditions.

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A modular open platform for systematic functional studies under physiological conditions.

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