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    Wyświetlanie 1-2 z 2
    Tytuł:
    Type III CRISPR complexes from Thermus thermophilus
    Autorzy:
    Szychowska, Marta
    Siwek, Wojciech
    Pawolski, Damian
    Kazrani, Asgar
    Pyrc, Krzysztof
    Bochtler, Matthias
    Powiązania:
    https://bibliotekanauki.pl/articles/1038832.pdf
    Data publikacji:
    2016
    Wydawca:
    Polskie Towarzystwo Biochemiczne
    Tematy:
    CRISPR-Cas
    endonuclease
    Csm
    Cmr
    Thermus thermophilus
    crRNA
    mass spectrometry (MS)
    chromatography
    Opis:
    Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.
    Źródło:
    Acta Biochimica Polonica; 2016, 63, 2; 377-386
    0001-527X
    Pojawia się w:
    Acta Biochimica Polonica
    Dostawca treści:
    Biblioteka Nauki
    Artykuł
    Tytuł:
    Pharmacological versus genetic inhibition of heme oxygenase-1 - the comparison of metalloporphyrins, shRNA and CRISPR/Cas9 system
    Autorzy:
    Mucha, Olga
    Podkalicka, Paulina
    Czarnek, Maria
    Biela, Anna
    Mieczkowski, Mateusz
    Kachamakova-Trojanowska, Neli
    Stepniewski, Jacek
    Jozkowicz, Alicja
    Dulak, Jozef
    Loboda, Agnieszka
    Powiązania:
    https://bibliotekanauki.pl/articles/1038402.pdf
    Data publikacji:
    2018
    Wydawca:
    Polskie Towarzystwo Biochemiczne
    Tematy:
    CRISPR/Cas9
    shRNA
    inhibitors
    heme oxygenase-1
    HO-1
    off-target
    Opis:
    Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may serve as a valuable therapy in various pathophysiological processes, including tumorigenesis. We compared the effect of chemical inhibitors - metalloporphyrins, with genetic tools - shRNA and CRISPR/Cas9 systems, to knock-down (KD)/knock-out (KO) HO-1 expression/activity. 293T cells were incubated with metalloporphyrins, tin and zinc protoporphyrins (SnPPIX and ZnPPIX, respectively) or were either transduced with lentiviral vectors encoding different shRNA sequences against HO-1 or were modified by CRISPR/Cas9 system targeting HMOX1. Metalloporphyrins decreased HO activity but concomitantly strongly induced HO-1 mRNA and protein in 293T cells. On the other hand, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. Nevertheless, silencing effect was much stronger when CRISPR/Cas9-mediated knock-out was performed. Most of the clones harboring mutations within HMOX1 locus did not express HO-1 protein and failed to increase bilirubin concentration after hemin stimulation. Furthermore, CRISPR/Cas9-mediated HO-1 depletion decreased 293T viability, growth, clonogenic potential and increased sensitivity to H2O2 treatment. In summary, we have shown that not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach.
    Źródło:
    Acta Biochimica Polonica; 2018, 65, 2; 277-286
    0001-527X
    Pojawia się w:
    Acta Biochimica Polonica
    Dostawca treści:
    Biblioteka Nauki
    Artykuł
      Wyświetlanie 1-2 z 2

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