Examinando por Autor "Duxan Arancibia"

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  • Miniatura
    Ítem
    Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    (2019) Sebastian Escobar Aguirre; Duxan Arancibia; Amanda Escorza; Cristián Bravo; María Estela Andrés; Pedro Zamorano; Víctor Martínez
    The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function
  • Miniatura
    Ítem
    Serine–Arginine Protein Kinase SRPK2 Modulates the Assembly of the Active Zone Sca olding Protein CAST1/ERC2
    (2019) Duxan Arancibia; Matias Lira; Yocelin Cruz; Daniela P. Barrera; Carolina Montenegro Venegas; Juan A. Godoy; Craig C. Garner; Nibaldo C. Inestrosa; Eckart D. Gundelfinger; Pedro Zamorano; VivianaI.Torres
    Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitmentoftheDrosophilaproteinBruchpilot(BRP),apartialhomologofCAST1/ERC2,ismodulated by the serine-arginine protein kinase (SRPK79D). Here, we demonstrate that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T and SH-SY5Y cells. More importantly, SRPK2 is present in brain synaptic fractions and synapses, suggesting that this protein kinase might control the level of self-aggregation of CAST1/ERC2 in synapses, and thereby modulate presynaptic assembly.