Allison Lynch

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Allison Lynch

• Fall 2006
• Gen 875 Class Presentation

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Purposes of RNAi analysis

• Functional genomic analysis
• Assign in vivo function to every gene by
  recording phenotypes
• Screening for genes involved in specific
  developmental or biological processes
• Enhancer screen

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Injecting RNAi in C. elegans

• Advantages
• Strong response
• Relatively quick
• Disadvantages
• Difficult technique
• Can cause artifacts

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Soaking RNAi in C. elegans

• Advantages
• Can select developmental stage of gene
  inactivation
• Relatively easy
• Disadvantages
• Weaker effect
• You have to starve the worms

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Feeding RNAi in C. elegans

• Advantages
• Very easy
• Very compatible approach
• Disadvantages
• Weaker effects
• Problems with bacterial libraries

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The phases of cytokinesis
Cytokinesis separation of cytoplasm by new
membrane
Initial Phase
Terminal Phase
Glotzer, 1997
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Why were they examining the cell cycle and
cytokinesis?
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Why is functional genomics important?

• The same DNA can yield different expression
  patterns
• Functional Genomics describe temporal and spatial
  gene function and interactions on a global scale
• Understand role of specific genes

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The Goals

• To identify all genes essential for mitotic cell
  division in a metazoan organism
• To bridge the gap between large-scale analyses
  and more in-depth studies
• i.e. devise a system to functionally annotate
  information that would allow for comparison to
  other datasets

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The Approach

• Use RNAi against genes in C. elegans
• C. elegans is a good model system because
• Invariant cell lineage, sequenced genome
• Time-lapse cytological analyses
• DIC microscopy
• The early embryo does not arrest in response to
  cell-cycle check mechanisms

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Generating a dsRNA collection

• 20,326 dsRNAs targeting 19,075 genes were
  designed, produced, and annotated
• Covered 98 of the genes in C. elegans

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The Method

• Injected either one or two dsRNAs into the gonads
  of C. elegans
• After 24 hours
• Filmed F1 progeny using DIC microscopy
• Assayed gross phenotypic differences in F1 progeny

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The Screen

• For 2093 genes of interest, injected dsRNA into
  worms and looked for phenotypes by
• DIC microscopy and
• progeny test
• Tested remaining genes in pairs using progeny
  test
• Lethal clones retested using DIC microscopy and
  progeny test
• Characterized clones with reproducible phenotypes

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Overall Results

• Of the 20,326 dsRNAs tested
• 1995 dsRNAs targeting 1668 genes had reproducible
  phenotype
• 661 genes affected the early embryo

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Thoroughness of Screen

• Compared RNAi results to previously published
  genetic analyses
• Initially identified 95 of these genes
• Compared RNAi results to previously published
  RNAi phenotypes
• DIC detection between 75 - 90

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Characterizing the RNAi phenotypes

• Scored each DIC recording for 45 distinct
  categories
• n 5 embryos, each from a different worm
• Grouped clones according to their phenotypic
  signature

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Characterizing the RNAi phenotypes

• Determined if phenotype classes corresponded to
  specific process
• Assign putative functions for additional genes in
  cluster

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Characterizing the RNAi phenotypes, continued
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So their second goal was to annotate their
results in a way that they could be applied to
in-depth studies of genes
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• http//www.worm.mpi-cbg.de/phenobank2/
• Can search for
• Genes and their phenotypes
• Defect map
• Primers used to generate dsRNA
• Also has links to other relevant databases