gene silencing plant

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Page 1: Gene silencing plant

presented by

Page 2: Gene silencing plant

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”

Page 3: Gene silencing plant

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

Page 4: Gene silencing plant

Methods for PTGSAntisense Oligonucleotides

Ribozymes

RNA Interference (RNAi)

Page 5: Gene silencing plant

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.

Page 6: Gene silencing plant

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.

Page 7: Gene silencing plant

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.

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Dicer

Page 9: Gene silencing plant

Applications• Cancer• Infectious diseases

Virus Bacteria

• Respiratory diseases• Neurodegenerative disorders

Huntington’s disease (HD) Amyotrophic lateral sclerosis (ALS)

Page 10: Gene silencing plant

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)

Page 11: Gene silencing plant

Virus• Human immunodeficiency virus (HIV) ->

chemokine receptor 5 (CR5)• Hepatitis B and C• Human papilloma virus ->E6• West Nile Virus• Tulane virus• Norovirus

Page 12: Gene silencing plant

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

Page 13: Gene silencing plant

Respiratory diseases• Asthma• Chronic obstructive pulmonary disease (COPD)• Cystic fibrosis

Cell hyperplasia Mucus hypersecretion Damaged lung tissue

growth factor (TGF)-αgrowth factor TGF-β

Page 14: Gene silencing plant

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

Page 15: Gene silencing plant

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

Page 16: Gene silencing plant

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.

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THANKYOU