Reverse Genetics Knockomics, Knockology'''

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Reverse Genetics(Knockomics, Knockology...

• Sequence to Phenotype to Function

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Forward vs. Reverse Genetics

• Treat thousands of organisms with a mutagen,
• random mutagenesis,
• Identify an individual with a phenotype of
  interest,
• Identify the gene.

• Treat thousands of organisms with a mutagen
  (usually),
• random mutagenesis,
• Identify an individual with a genotype of
  interest,
• Identify the phenotype.

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Homologous Recombination

• the replacement of a gene with an exogenous gene
  through equal crossing over,

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Homologous Recombination Range

• Yes...
• mice, many well characterized mammalian cells,
• bacteria,
• yeast, (remember the bar code deletion project),
• No (maybe)...
• C. elegans (no),
• Arabidopsis (done once, not repeated),
• Drosophila (shown in principle, not repeated),
• the rest?

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Over Expression Studies

• Make a gene construct with,
• Structural Gene,
• Active promoter (often from a virus promoter),
• Marker gene to be able to determine
  transformation.

• Expect,
• Higher levels of protein,
• Gene-dosage phenotypes,
• Glorious publication.

Frequent Results no protein produced, scorn from
senior scientists.
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Anti-Sense Studies

• Another good idea use a transgene with the
  coding sequence reversed...

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Huh?
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Expected Results

• Low, to no detectable transcript,
• Low, to no protein products,
• Glorious publication detailing gene function.

• Actual Results (Wacky)
• Phenotypes ranged from death to
  over-expression,
• Transcript levels were also extremely variable,
• Scorn from senior scientists.

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Co-suppression(RNA turnover)

• T-DNA insertion can happen more than once per
  plant,
• Hard-headed, high self-esteem plant scientists
  published results indicating that...
• transgene expression often decreased as the copy
  number of transgene increased,
• Plants with more than one over-expression, or
  anti-sense construct, seemed to have the least
  transgene expression.

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Modes

• ...Transcriptional Gene Silencing (TGS),
• RNA influences the methylation of promoters,
• ...Post-Translational Gene Silencing (PTGS),
• appears to involve the specific degradation of
  mRNA via a double-stranded RNA intermediate,
  dsRNA.

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Co-Suppression as Reverse Genetics

• Plant scientists began using over-expression as a
  means to turn-off gene expression,
• Strangely, over-expressed genes were present on
  Northern blots as very small molecules,
• i.e. transcripts that should have been kbs in
  length, were present as 20-mers on Northerns.

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RNAi(RNA interference)

• ...while attempting to do anti-sense KO of gene
  expression in C. elegans, Guo and Kemphues, Cell
  81, 611 (1995) observed that sense and anti-sense
  strands worked equally,
• in an anti-sense experiment, a gene is
  constructed so that it produces a complementary
  strand to an expressed transcript,
• the goal is to complement, thus inactivate the
  mRNA.
• ...following up, they found that dsRNA worked at
  least an order of magnitude better that either
  sense or anti-sense strands.

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dsRNA
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Genome Surveillance

• ...epigenetic control of gene expression is now
  considered a component of normal development,
• stable repression of genes not required by
  specific cell types,
• defense against viruses,
• control of transposable elements,
• others?

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dsRNA Delivery

• ...dsRNA can move across cell boundaries,
• through the gut of C. elgans,
• injected,
• ingested, E. coli expressing a dsRNA,
• soaking,
• (through the vascular system of plants),
• ...or delivered as a heritable transgene.

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(No Transcript)
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It Works

• Arabidopsis

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Disruption Mutations

• ...mutations that disrupt genes so that no
  functional protein is produced,
• insertion mutations,
• deletion mutations (C. elegans).

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Transposable Elements

• a segment of DNA that can move to, or move a
  copy of itself to another locus on the same or a
  different chromosome (hopping DNA),
• may be a single insertion sequence, or a more
  complex structure (transposon) consisting of two
  insertion sequences and one or more intervening
  genes,
• may be engineered to include marker genes,
  promoters, etc.

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Transposable Elements
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Transposition
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Transposons
Two transposable elements flanking other DNA, the
whole complex hops.
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Transposons

• Pluses
• organisms do most of the work,
• local transposition,
• potential excision returns wt gene,
• Minuses
• hot spots,
• unstable,
• may require specific breeding to
  mobilize/stabilize transposition, requiring
  extensive genotyping.

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T-DNA Insertion Mutants

• PCR based approach to identify plants with
  foreign DNA inserted into genes of interest
• Agrobacterium Transformed Plants,
• Agrobacterium transfers genetic material (T-DNA)
  into the plant genome during the course of
  pathogenesis,
• use primers specific for the target gene and
  primers for the T-DNA insert,
• isolate single plants from a large pool.

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Agrobacterium tumefaciens

• Natural soil bacterium that infects plants,
• hosts 160 Genera,
• families gt 60,
• infection results tumors, poor growth, low
  yield.

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Natural Infection

• Agrobacterium has a tumor inducing plasmid (Ti
  Plasmid) that upon infection, is transferred to
  the plant cell,
• Ti Plasmid integrates into the plants genome,
• cancer-like cell growth is induced,
• genes coding for biosynthetic pathways that
  produce nutrients specific for Agrobacterium are
  transcribed and translated.

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T-DNA

• Transfer DNA disarmed Ti Plasmid,
• genes that induce cancer-like growth are removed,
• genes that code for Agrobacterium nutrients are
  removed,
• T-DNA is then re-introduced into Agrobacterium,
  Agro infects the plants, and the T-DNA inserts
  into the plant genome.

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Ti Plasmid
wt plant chromosome
hormone genes
opaline
virulence genes
Ti Plasmid
nopaline
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T-DNA
marker gene(s)
plant promoter
virulence genes
Construct T-DNA
if the T-DNA lands in a gene, the gene is
disrupted.
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Finding Random Insertion Mutants

• Use PCR based approach to identify sequence with
  foreign DNA inserted into genes of interest.

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PCR Strategy

• Polymerase Chain Reaction (PCR),
• with oligonucleotide primers with homology to the
  5 and 3 ends of your gene, amplify the DNA
  sequence between the primers.

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Reaction
3
Your gene
Product
Your gene amplified
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Reverse Genetic PCR Strategy
Reaction
Product none.
T-DNA
Reaction
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Probability of Finding an Insert in a Specific
Gene
p 1-(1-f)n
thousands of inserts
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Transformation

• Construct, or borrow an unarmed T-DNA plasmid,
• transform it into a virulent race of
  agrobacterium.
• grow the bacterium, under selection (T-DNA
  specific),
• centrifuge cells into pellet,
• re-suspend in a really strong detergent,
• Grow T0 (wt) plants until they are ready to
  flower,
• Dip the plants in the agro/detergent medium.

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One Line

• Collect T1 seeds,
• Grow on plant selection media,
• Plants that grow contain a T-DNA.

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DNA Pooling
Maintain lines as pools of seed.
Seeds (9)
Germinate and grow seeds in liquid culture.
Seedlings (225)
Extract DNA,
DNA (225)
Super Pool DNA,
Super Pools (2000)
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2
3
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7etc.
PCR Screen
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PCR Strategy
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T-DNA Insertion Confirmation
G
wt

• Blot gel and hybridize with a WT probe.
• Band isolate and cycle sequence PCR fragment.

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Sequence T-DNA Insertion Sites
Sequence using the PCR primer from the T-DNA
sequence.

• T-DNA
  Unknown ATPase
• AHA1 \\GCAATGTGTTATTAAGTTGTCT ---
  (CCC...15...AAA) --- GAAAATTTTCGCCACTGGAAAT//
• AHA2 \\TGTCTAAGCGTCAATTTGTTTA ---
  (ATG...38...TAC) --- ACCTGCTCAGGAGCACCTTTAC//
• AHA4 \\AAGGGATCTTTTTGGAATGCTG
  -------------------------------
  GAACTAGTTTCAGAAGTACATG//
• AHA5 \\TCTAAGCGTCAATTTGTTTACA
  --------------------------------
  TGCCCTTTTAATGTATTTATCT//
• AHA10 \\TTTGTTTACACCACAATATATC ----- (GTCCGGGA)
  ----- TGTGATTTTTCATTTCTTTAGC//

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Next Generation Knockology

• Index Tagged Knockouts,
• Usually via TAIL PCR,
• Thermal Asymmetric Interlaced PCR

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Reverse Genetics
Function
Sequence
Gene Disruption
Phenotype
ESTS GENOMIC DNA
Homologous recombination Overexpression Anti-sense
RNAi Tagnology Transposons T-DNA
Genetics Biochemistry Physiology
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Phenomics

• The science of identifying meaningful
  phenotypes in reverse genetic studies.

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Wednesday