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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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
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
Genome screen by feeding wormsGenome screen by feeding wormswith dsRNA expressing with dsRNA expressing E. coliE. coli
Tuschl Tuschl 2003 2003 NatureNature
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
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
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
2.TILLING (Targeting Induced Local Lesions IN Genomes)
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
Homologous recombination and gene knock-out in yeast and mouse
Fig. A.8
Fig. A.7
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