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Serial cloner cohesive ends
Serial cloner cohesive ends










Mohr S, Bakal C, Perrimon N (2010) Genomic screening with RNAi: results and challenges. CRC Press, Boca Ratonįalschlehner C, Steinbrink S, Erdmann G et al (2010) High-throughput RNAi screening to dissect cellular pathways: a how-to guide. In: Sohail M (ed) Gene silencing by RNA interference. Morris JC, Wang Z, Motyka S et al (2004) An RNAi-based genomic library for forward genetics in the African Trypanosome. Artif Intell Med 35:61–73Ĭelotto AM, Lee JW, Graveley BR (2005) Exon-specific RNA interference: a tool to determine the functional relevance of proteins encoded by alternatively spliced mRNAs. Zhao W, Fanning ML, Lane T (2005) Efficient RNAi-based gene family knockdown via set cover optimization. Ory JJ, Griffith CL, Doering TL (2004) An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter. Ruff JA, Lodge JK, Baker LG (2009) Three galactose inducible promoters for use in C. Wickes BL, Edman JC (1995) The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter. Gene 232:155–163Ĭox GM, Rude TH, Dykstra CC et al (1995) The actin gene from Cryptococcus neoformans: structure and phylogenetic analysis. Varma A, Kwon-Chung KJ (1999) Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene and the use of its promoter for heterologous expression in Cryptococcus neoformans, a human pathogen. Biochem Biophys Res Commun 390:983–988įu J, Hettler E, Wickes BL (2006) Split marker transformation increases homologous integration frequency in Cryptococcus neoformans. Kim MS, Kim SY, Yoon JK et al (2009) An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Goins CL, Gerik KJ, Lodge JK (2006) Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes. Reilly MC, Levery SB, Castle SA et al (2009) A novel xylosylphosphotransferase activity discovered in Cryptococcus neoformans. Hu G, Hacham M, Waterman SR et al (2008) PI3K signaling of autophagy is required for starvation tolerance and virulence of Cryptococcus neoformans. Reese AJ, Doering TL (2003) Cell wall alpha-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Panepinto J, Liu L, Ramos J et al (2005) The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans. Panepinto J, Komperda K, Frases S et al (2009) Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans. Liu H, Cottrell TR, Pierini LM et al (2002) RNA interference in the pathogenic fungus Cryptococcus neoformans. Gorlach JM, McDade HC, Perfect JR et al (2002) Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy. Curr Opin Microbiol 11:494–502ĭe Backer MD, Raponi M, Arndt GM (2002) RNA-mediated gene silencing in non-pathogenic and pathogenic fungi. Nakayashiki H, Nguyen QB (2008) RNA interference: roles in fungal biology. Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Malone CD, Hannon GJ (2009) Small RNAs as guardians of the genome. Verdel A, Vavasseur A, Le Gorrec M et al (2009) Common themes in siRNA-mediated epigenetic silencing pathways. Nakayashiki H, Kadotani N, Mayama S (2006) Evolution and diversification of RNA silencing proteins in fungi. neoformans by electroporation, screening clones for RNAi-related phenotypes, and evaluating the efficacy and specificity of gene silencing by RNAi. neoformans and describe the steps for subcloning into either vector, transforming C. We provide guidelines for selecting a suitable interfering sequence to trigger RNAi in C. Both vectors are designed to co-silence a “sentinel” gene with an easily scored phenotype to help identify clones in which RNAi is most effective. The pIBB103 vector relies on convergent, inducible GAL7 promoters to independently drive the synthesis of the sense and antisense strands of the interfering sequence these strands anneal to form the initiating dsRNA molecule. The pFrame vector utilizes the ACT1 promoter to enable the constitutive synthesis of hairpin RNA (hpRNA), the stem of which constitutes the dsRNA trigger. Here, we describe two plasmid-based strategies we have developed for RNAi in Cryptococcus neoformans. The process relies on double-stranded RNA (dsRNA) to target complementary messenger RNA for degradation. RNA interference (RNAi) is an experimental technique used to suppress individual gene expression in eukaryotic cells in a sequence-dependent manner.












Serial cloner cohesive ends