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Reverse Genetics: interference RNA Mitchell Bush Mimosa Chau Tayler Fluharty Summary • Use RNAi to circumvent gene replacement difficulties in Cryptococcus neoformans. • Focus on two genes of known phenotype: CAP59 Codes for product required for polysaccharide capsule. Polysaccharide capsule required for virulence. ADE2 Codes for phosphoribosylaminoimidazole carboxylase. Loss of AIR carboxylase results in adenine intermediates. General Mechanism of RNAi Double stranded RNA is cleaved into smaller fragments of about 22 bp by Dicer where degradation of corresponding mRNA is degraded with assistance of RISC protein complex. Interference can persist for many round of cell division and can be passed on to next generation. http://nobelprize.org/nobel_prizes/medicine/laureates/20 06/adv.html Cryptococcus neoformans • Encapsulated fungal pathogen. • Haploid yeast, can undergo sexual cycle • Causes infections in immunodeficient individuals. • 24 Mb genome. • High frequency of nonhomologous recombination. • Virulence factor = polysaccharides capsule. Cell strains • Wild-type serotype D strain B4500 (transformed with control plasmid lacking RNAi construct) • cap59 strain TYCC33 (CAP59 knocked-out/positive control) • ura5 strain JEC43 (used for two type of selection during experiment) Hairpin Construct • 500 base Inverted Repeat(s) • 250 base spacer of GFP CAP59 encodes product required for synthesis of polysaccharide capsule which gives cells a “shiny” appearance CAP59i cells lack polysaccharide capsule (note exception) ADE2 encodes for phosphoribosylaminoimidazole carboxylase ADE2i cells accumulate adenine biosynthetic intermediates, resulting in pink colonies. DIC Fluorescence DIC Fluorescence 5-FOA is converted to toxic product in presence of RNAi construct (pCAP59i/ pADE2i) Therefore 5-FOA selects against organisms containing the RNAi construct. Mutant phenotype caused by exogenous DNA The ADE2 gene in the genome was not disrupted. RNAi is dependent on Transcription Made plasmid that lacked promoter for CAP59i. No transcription=No altered phenotype Summary of Results • The effect of RNAi is variable. • Different genes have different threshold for phenotypic expression. • CAP59 has a lower threshold than ADE2 Range of interference Pro study of essential genes-if complete inhibition were lethal Con Investigation of novel genes requires 2phases of study: level of mRNA and analysis of transformants exhibiting interference. In the Works Fight a virus with RNAi: Foot and mouth disease virus (FMDV) • Data base search for highly conserved regions of FMDVThree 21 base sequences found. • Silence out one at a time, then all three Viral inhibition (%) Cons 7 80 Cons 8 92 Cons 9 87 mix >98 Three potential mechanisms to fight HIV: 1) CD4 silencing resulted in reduced HIV-1 entry, but use may be limited due to normal function of CD4 2) Blocking HIV genomic RNA with p24 siRNA resulted in reduced viral mRNA (CCR5 is potential future target lack of this receptor does not disrupt regular cell function) 3) Blocking HIV transcript with p24 siRNA inhibits HIV1 replication RNAi and cancer • Metastatic melanoma – Genasense • Antisense oligonucleotide target antiapoptotic gene BCL2. • Bcr-Abl oncoprotein (chronic myelogenous leukaemia ) p210 targeted with RNAi. • Delivery proves to be a challenge. • Delivery agents such as cationic lipids or polymers. Problem = toxic. Delivery methods • Oligonucleotides/siRNA delivered via endocytosis. • CTLs, cell targeting ligands – increases target cell interactions. • CPP primarily cell penetrating peptides enhance transmembrane permeation – associate with oligonucletide, binds to cell surface glycosaminoglycans. “Significant strides have been made, but the issue has not been fully resolved.” -Juliano, Et. al References Daneholt, B. (2006). The Nobel Prize in Physiology or Medicine 2006. Retrieved March 8, 2009, from Nobelprize.org: http://nobelprize.org/nobel_prizes/medicine/laureates/2006/adv.html Hannon, G. J., & Rossi, J. J. (2004). Unlocking the potential of the human genome with RNA interference. Nature , 371-378. Juliano, R., Alam, R., Dixit, V., & Kang, H. (2008). Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Research , 1-14. Kahana, R., Kuznetzova, L., Rogel, A., Shemes, M., Hai, D., Yadin, H., et al. (2004). Inhibition of foot-and-mouth disease virus replication by smal interfering RNA. Journal of General Virology , 3212-3217. Liu, H., Cottrell, T. R., Pierini, L. M., Goldman, W. E., & Doering, T. L. (2001). RNA Interference in the Pathogenic Fungus Cryptococcus neoformans. Genetics Society of America , 463-470. Novina, C. D., Murray, M. F., Dykxhoorn, D. M., Beresford, P. J., Riess, J., Lee, S.-K., et al. (2002). siRNA-directed inhibition of HIV-1 infection. Nature Medicine , 681-686. Applause