Lecture 15: Functional Genomics IIscience.umd.edu/classroom/BSCI410-Liu/BSCI410/07... · Lecture...
Transcript of Lecture 15: Functional Genomics IIscience.umd.edu/classroom/BSCI410-Liu/BSCI410/07... · Lecture...
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Lecture 15: Functional Genomics II
– High-throughput RNAi screens– High-throughput insertional/chemical screens– Homologous recombination (yeast and mouse)- Other methods in discerning gene function
– Activation tagging– Enhancer trapping– Modifier screens (enhancer and suppressors),- Yeast Two Hybrid Assay
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RNAi all genes on chromosome IIIRNAi all genes on chromosome III--GGöönczynczy, et al, 2000, et al, 2000
• Goal: In C. elegans, determinefunction of all 2,300 genes onchromosome III
• RNAi constructs made for eachgene
• Worms microinjected with double-stranded RNA
• Videos made of embryonicphenotypes
T7 T3
add T3 + T7polymerase
predicted ORF
construct withpromoter sequence
dsRNA
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Genome screen by feeding wormsGenome screen by feeding wormswith dsRNA expressing with dsRNA expressing E. coliE. coli
Tuschl Tuschl 2003 2003 NatureNature
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Identify gene function by Identify gene function by insertionalinsertionalor chemical mutagenesisor chemical mutagenesis
1) T-DNA or transposon insertionsand PCR-based screens
2) Arabidopsis Tilling project
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Gene X
NPTII
FR
LB
RB
PCR products:
1. Screen for T-DNA (or Ds) insertion in specific genes
Screening pools (p1-p5)
p1 p2 p3 p4 p5 1kb ladder p1 p2 p p4 p5
LB/F RB/R
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Data-base searches for T-DNA insertions in the genes of interests
Gene X
LB
RB
Enzyme digestion
Ligase
PCR Sequence database
Salk Institute Genomic Group (http://signal.salk.edu/cgi-bin/tdnaexpress)
LEUNIG="At4g32550SEU=At1g43850
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2.TILLING (Targeting Induced Local Lesions IN Genomes)
Arabidopsis Tilling website:http://tilling.fhcrc.org:9366/
EMSIsolated DNA
poolGene-specific
primersPCRheat & coolCEL I
Denature
1 2 3 4 5 6 7 8 9 10
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Classical mutagenesisClassical mutagenesis vs vs. RNAi. RNAi
• Diversity of mutations– (point mutations, deletions,
inversions, etc.)
• Heritable, stable, and quantitative
• Saturating the genome requireshitting multiple genes repeatedly
• Give each gene equal attention
• High throughput
• Reasonably equivalent disruption ofeach locus
• “Mutations” automatically mapped
• Not heritable
• Doesn’t generate full depletion oftarget RNA
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Homologous recombination and gene knock-out in yeast and mouse
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Fig. A.8
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Fig. A.7
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Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst.The stem cells can become any tissue in the body, excluding a placenta.
Transgenic mouse depends on ES (embryonic stem) cells
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Knockout Mice Targeting ConstructsKnockout Mice Targeting Constructsfor Homologous Recombinationfor Homologous Recombination
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Transgenic MiceTransgenic Mice~Generation of Knockouts~~Generation of Knockouts~
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Activation tagging in Activation tagging in ArabidopsisArabidopsis
• Strong constitutive viral promoter– CaMV 35S
• Inserted randomly in genome– With T-DNA
• When inserts are near a gene promoter, thefollowing results occur:
– Activation– Constitutive expression
• Because many genes are expressed in specificcells or tissues, activation in all tissues canresult in abnormal phenotypes
gene Xconstitutive expression of gene X
35S enhancer
35S enhancer
T-DNA vector
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eyelesseyeless gene gene
Wild type
Ectopic expression of eyeless
eyeless
Loss-of-function vs. gain-of-function usually give opposite phenotypes
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Activation taggingActivation tagging• A variation on insertional mutagenesis
– Makes gain-of-function mutations instead ofloss-of-function mutations
– An insertion that carries a strong constitutive promoteror enhancer
• Potential to identify gene function not detectable through loss-of-function screens– Useful for the following cases:
• Functionally redundant genes• Genes required for viability
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Gene X
GUS
Minimal promoter
Enhancer of gene X
T-DNA
Dr. Tom Jack’s websitehttp://www.dartmouth.edu/~tjack/
Enhancer trapping in Arabidopsis
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ap1-1 ap1-1 cal-1
ap1-1 cal-1
Modifier screen example
cal-1: wild-type lookingap1-1: flower mutantap1-1 cal-1: cauliflower
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Yeast Two Hybrid (Y2H) Assay to test interaction between two proteins
GAL4DB
GAL4AD
X Y
HIS3 or lacZ5XUAS
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LUFS
LUFS+Q-rich
Q-rich+WD
Positive Control
LUFS+Q-rich+WD
Yeast Two -Hybrid Assay for Interaction Between LUG and SEU
SEU-GAL4-ADVS.
Q-rich (89-184, 449-470)
7 WDLUFS Q-rich (89-184, 449-470)
7 WDQ-rich (89-184, 449-470)
LUFS
LUFS
LUG-GAL4-DB constructs
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Use NCBI’s Books to search for:
Yeast two hybrid assayActivation taggingEnhancer trapEtc.