gene silencing plant
TRANSCRIPT
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What is Gene Silencing?• “Turning off" a gene• This process happens naturally• Occurs at a transcriptional or a post-
transcriptional level• An important laboratory technique• Epigenetic regulation of gene expression• Don’t confuse it “gene knockout”
Types
Transcriptional Post-transcriptional Meiotic
a) Genomic Imprinting
b) Paramutationc) Transposon
silencing d) Transgene
silencinge) Position effectf) RNA-directed
DNA methylation
a) RNA interferenceb) Nonsense
mediated decay
a) Transvectionb) Meiotic
silencing of unpaired DNA
Methods for PTGSAntisense Oligonucleotides
Ribozymes
RNA Interference (RNAi)
Antisense Oligonucleotides• Paul Zamecnik and Mary Stephenson (1978)• Short nucleic acid fragments• Bind to complementary target mRNA molecules• Mechanisms-
RNase H-dependent Steric blocking mechanism
• 80% to 95% decrease in the protein and mRNA expression.
Ribozymes• Sidney Altman and Thomas Cech (1989)• Cleave mRNA molecules• Types-
Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs group I and group II ribozymes RNase P ribozyme
• Similar mechanism as protein ribonucleases• Sequence-specific cleavage of target mRNA molecules.
RNA Interference (RNAi)• Andrew Fire and Craig Mello (1998)• Entry of double-stranded RNA (dsRNA)• Small double-stranded fragments by Dicer
Small interfering RNAs (siRNA) MicroRNA (miRNA)
• RNAi induced silencing complex (RISC)-> Argonaute proteins• "guide" strand, binds to RISC• “passenger” strand degraded• Cleavage or translational repression of the mRNA molecules
makes the genes inactive.
Dicer
Applications• Cancer• Infectious diseases
Virus Bacteria
• Respiratory diseases• Neurodegenerative disorders
Huntington’s disease (HD) Amyotrophic lateral sclerosis (ALS)
Cancer• Chronic myelogenous leukemia (CML) -> BCR-
ABL• Mutant tumor suppressor p53 molecules• Chemokine receptor 4 (CR4)• Antiapoptotic proteins (clusterin and survivin)• Colon adenocarcinoma (B-catenin)
Virus• Human immunodeficiency virus (HIV) ->
chemokine receptor 5 (CR5)• Hepatitis B and C• Human papilloma virus ->E6• West Nile Virus• Tulane virus• Norovirus
Bacteria• Host genes
Involved in immune response caused by infection Involved in mediating the entry of bacteria into
cells• Lipopolysaccharide (LPS) -> tumor necrosis
factor α (TNFα)• Psueomonas aeruginosa -> caveolin-2 (CAV2)
gene
Respiratory diseases• Asthma• Chronic obstructive pulmonary disease (COPD)• Cystic fibrosis
Cell hyperplasia Mucus hypersecretion Damaged lung tissue
growth factor (TGF)-αgrowth factor TGF-β
Huntington’s disease• Mutation in the huntingtin gene• Mutated huntingtin protein• Motor, cognitive, and behavioral deficits• Allele specific gene silencing
Antisense oligonucleotides-single nucleotide polymorphism (SNPs)
• Non-allele specific gene silencing Normal and mutated huntingtin proteins
Amyotrophic lateral sclerosis• Lou Gehrig’s disease• Brain and spinal cord; motor neurons
degenerate, death• Mutations in the Cu/Zn superoxide dismutase
(SOD1)• Allele-specific gene silencing
ConclusionDelivery and SpecificityFor neurodegenerative disorders, gene silencing
molecules must be injected directly or by pumpsViral vectors can be usedMore efficient methods to deliver and develop
specific gene silencing therapeuticsSafe and effective.
References http://en.wikipedia.org/wiki/Gene_silencing Kole, R; Krainer, AR; Altman, S (Jan 20, 2012). "RNA therapeutics:
beyond RNA interference and antisense oligonucleotides.". Nature reviews. Drug discovery 11 (2): 12540. doi:10.1038/nrd3625. PMID 22262036
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