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    Gene Knockdown: MBB156: Analysis Session 8 Bay 4

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    kirandedhia
    1) Gene knockdown is a technique that allows for reduced expression of one or more genes, which can be done through genetic modification or treatment with a complementary oligonucleotide. 2) It is used in research to learn more about genes that have been sequenced but have unknown functions by studying the differences between normal and knockdown organisms. 3) Techniques for knockdown include DNA-binding oligonucleotides that prevent transcription and RNA-binding oligonucleotides like siRNA and miRNA that promote mRNA degradation to reduce protein translation.

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    Gene Knockdown: MBB156: Analysis Session 8 Bay 4

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    kirandedhia
    87 views11 pages
    1) Gene knockdown is a technique that allows for reduced expression of one or more genes, which can be done through genetic modification or treatment with a complementary oligonucleotide. 2) It is used in research to learn more about genes that have been sequenced but have unknown functions by studying the differences between normal and knockdown organisms. 3) Techniques for knockdown include DNA-binding oligonucleotides that prevent transcription and RNA-binding oligonucleotides like siRNA and miRNA that promote mRNA degradation to reduce protein translation.

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    Gene Knockdown

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    1) Gene knockdown is a technique that allows for reduced expression of one or more genes, which can be done through genetic modification or treatment with a complementary oligonucleotide. 2) It is used in research to learn more about genes that have been sequenced but have unknown functions by studying the differences between normal and knockdown organisms. 3) Techniques for knockdown include DNA-binding oligonucleotides that prevent transcription and RNA-binding oligonucleotides like siRNA and miRNA that promote mRNA degradation to reduce protein translation.

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    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    87 views11 pages

    Gene Knockdown: MBB156: Analysis Session 8 Bay 4

    Uploaded by

    kirandedhia
    1) Gene knockdown is a technique that allows for reduced expression of one or more genes, which can be done through genetic modification or treatment with a complementary oligonucleotide. 2) It is used in research to learn more about genes that have been sequenced but have unknown functions by studying the differences between normal and knockdown organisms. 3) Techniques for knockdown include DNA-binding oligonucleotides that prevent transcription and RNA-binding oligonucleotides like siRNA and miRNA that promote mRNA degradation to reduce protein translation.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 11

    Gene Knockdown

    MBB156: Analysis Session 8


    Bay 4

    Created By Kiran Dedhia, Mkuzi,


    Nkhata, Charlotte Young, Max
    Hebditch, Robyn Bradbury,
    Olympia Gianfrancesco, James
    Crowe and???

    Presented By Kiran Dedhia


    Introduction

    • Gene knockdown is a technique which allows the


    expression of one or more genes to be reduced.
    • This can be done in two different ways.
    • Genetic Modification.
    • This involves a permanent change in the DNA leading to
    permanently reduced gene expression.
    • Treatment with a specific reagent.
    • A short piece of DNA or RNA with a sequence complementary
    to the gene or mRNA transcript.
    • This leads to a temporary reduction in expression
    • Creating a transient knockdown
    •One Key Question
    Why do we use gene knockdown

    So far only it has only been used for research


    1

    To learn more about a gene that has been


    2
    sequenced but with an unknown function
    Difference between normal organism and
    3
    knockdown organism is investigated
    To study the effect of decreased expression of
    4
    a gene in an organism
    It is easier to knockdown rather than knockout
    5
    a gene
    •Transient Knockdown
    • Binding of oligonucleotide
    causes decreaded
    expression
    • DNA primer with
    complementary sequence
    to gene blocks translation
    • Prevention of translation is
    achieved by RNA
    interference (RNAi)
    • RNAi achieved through
    miRNA and siRNA
    • Complementary binding
    with the target mRNA
    promotes degradation of
    mRNA and blocks pre-
    mRNA splicing.
    •DNA binding of oligonucleotides
    3 types of DNA bindings
    • Minor groove binding (A)
    • Polyamides bind at minor
    groove of DNA
    • Major groove binding (C)
    • Short DNA fragment that
    interact like Hoogsteen base
    pairs and form triple stranded
    DNA
    • PNA (peptide nucleic acid)
    binding (B)
    • Sugar-phosphate backbone
    is replaced by 2-aminoethyl
    glycine carbonyl
    • All prevent binding of RNA
    polymerase, transcription
    factors or induce gene
    specific damage to DNA
    •RNA binding of oligonucleotides
    2 types of RNA used
    • siRNA (small interfering RNA)
    • dsRNAs get broken down into smaller
    fragments by Dicer, an endonuclease,
    after insertion. These fragments are called
    siRNA
    • Weak hydrogen bonds break and strands
    separate
    • siRNA bind to RISC,(RNAi silencing
    complex) a ribonucleoprotein, to form
    siRNA/RISC complex
    • mRNA associates with complementary
    siRNA, RISC catalyses cleavage of mRNA
    • mRNA fragments degraded by other
    nucleases

    • miRNA (micro RNA)


    • Gets cleaved by nucleases into mature
    form
    • Strands separate
    • Anti-sense miRNAstrand (non-coding) bind
    to mRNA
    • Binding prevents RNA polymerase from
    reading mRNA thus preventing translation
    •Knockdown organisms

    • Organisms in which the expression


    of a gene has been reduced

    • Knockdown techniques appear to


    be easier to carry out than
    complete knockout technique

    • Organisms are made for


    investigation into the function of a
    particular gene, by injecting an
    embryonic cell with siRNAs which
    interfere with transcription
    •Reverse Genetics
    INTRODUCTION TECHNIQUES OF REVERSE GENETICS

    • Reverse genetics is the DIRECTED DELETIONS AND


    opposite of classical POINT MUTATIONS
    genetics, or ‘forward • Site‐directed mutagenisis is
    genetics’ in that the gene used to change regulatory
    sequence is identified first regions in a promoter site or
    and a phenotype associated make subtle changes to
    with this gene is then worked codon sequences to the ORF
    out. to see which amino acid
    • Scientists use reverse genetics residues are important to
    to connect a given genetic protein function.
    sequence with specific • This technique can also be
    effects on the organism. used to make gene
    non‐functional, these are
    called null alleles.
    Techniques
    • Gene silencing
    • Using double stranded RNA also known
    as RNA intereference (RNAi)
    • Creates a specific knockout without actually mutating the
    DNA of interest.
    • Acts by directing cellular systems to degrade mRNA
    • Morpholino Oligosaccharides
    • Bind to block access to target mRNA without requiring the
    activity of cellular proteins
    • Effective in systems
    Techniques (cont…)
    • Interference using Transgenes
    • Molecular genetic approach
    • Organisms that over expresses a normal gene
    of interest OR
    • Organisms that over expresses a mutant form of a gene that
    interferes with wild type function
    • Dominant negative interaction
    Future aspects

    Post-translational silencing of gene expression


    is a powerful molecular tool for stem cell
    research to study gene function,
    biological pathways and physiology of
    disease. Gene silencing via siRNA enables
    several applications:

    Developmental pathway screening to


    determine the gene function and identify
    transcription factors that regulate self-renewal,
    proliferation, differentiation and apoptosis.

    Directed differentiation of embryonic or tissue-


    specific stem cells by silencing genes of
    pluripotency maintenance, such as
    Oct 4 or Nanog to induce early differentiation
    and lineage determination.

    Reprogram or dedifferentiate tissue specific


    stem cells to repair damaged tissues or organs.

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