lecture 11. functional genomics at the level of the whole organism: genomic approaches to biology
TRANSCRIPT
Lecture 11. Functional Genomicsat the Level of the Whole Organism:
Genomic Approaches to Biology
One goal of Functional Genomics is todefine the function of all genes, and to define how genes interact to form morecomplicated networks responsible forbiological processes.
Ways we have discussed to accomplish this:
1) Expression Clustering (either at RNA or Protein Levels)2) Protein:Protein Interaction Maps (both in vivo and in vitro)3) Predictions based on Protein Structure(protein structure=function)
However, the function and interaction of genes must be tested in the ENTIRE ORGANISM
Goals of Functional Genomics:1)DNA2)RNA3) Protein4) Whole organism5) Society
Lander, E. 1996. The New Genomics: Global Views of Biology. Science 274: 536-539.
4. Whole organism Genetic tools for manipulating cell circuitry a) systematic knockout and mutation of genes:
both stable and conditional b) transgenic studies: overexpression of gene products c) redesigning of cellular circuits (e.g., drosophila gal4 enhancer traps)
Model Systems are especially important.
4100 genes 6000 genes 18,000 genes 14,000 genes
35-70,000 genes?
50 genes
Importance of MODEL SYSTEMS in GenomicsGenome Size and Gene Number in Model Organisms and Man
Topics for Today’s Lecture:
1. Systematic mutation of genes in YEAST to determine gene function
2. Targeted knockouts and conditional knockouts, and 5’ gene Traps in MICE
6000 GENES
Targeted Deletions in Yeast
Functional Profiling of theSaccharomyces cerevisiae Genome
click for: [abstract] [supplemental data]
Guri Giaever1, Angela M. Chu, Li Ni, Carla Connelly, Linda Riles, Steeve Véronneau, Sally Dow, Ankuta Lucau-Danila, Keith Anderson, Bruno André, Adam P. Arkin, Anna Astromoff, Mohamed el Bakkoury, Rhonda Bangham, Rocio Benito, Sophie Brachat, Stefano Campanaro, Matt Curtiss, Karen Davis, Adam Deutschbauer, Karl-Dieter Entian, Patrick Flaherty, Francoise Foury, David J. Garfinkel, Mark Gerstein, Deanna Gotte, Ulrich Güldener, Johannes H. Hegemann, Svenja Hempel, Zelek Herman, Daniel F. Jaramillo, Diane E. Kelly, Steven L. Kelly, Peter Kötter, Darlene LaBonte, David D. Lamb, Ning Lan, Hong Liang, Hong Liao, Lucy Liu, Chuanyun Luo, Marc Lussier, Rong Mao, Patrice Menard, Siew Loon Ooi, Jose L. Revuelta, Christopher J. Roberts, Matthias Rose, Petra Ross-Macdonald, Bart Scherens, Greg Schimmack, Brenda Shafer, Daniel D. Shoemaker, Sharon Sookhai-Mahadeo, Reginald K. Storms, Jeffrey N. Strathern, Giorgio Valle, Marleen Voet, Guido Volckaert, Ching-Yun Wang, Teresa R. Ward, Julie Wilhelmy, Elizabeth A. Winzeler, Yonghong Yang, Grace Yen, Elaine Youngman, Kexin Yu, Howard Bussey, Jef D. Boeke, Michael Snyder, Peter Philippsen13,
Ronald W. Davis1,2 & Mark Johnston5
http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html
Transposon-Mediated Random Mutation Strategy
CRE/LoxP Recombination System
http://ygac.med.yale.edu/triples/triples.htm
Random Mutagenesis Strategy
Phenotypic Macroarray Analysis
Measure Growth of Mutantsin 96 well format
GrowthConditions
Cluster Analysis of the Data
Columns:Growth Conditions
Rows: Various Mutants
After CRE expression: study protein localization byimmunohistochemistry
2. Gene Targeting in Mouse:Deletions and Conditional Deletion using CRE/loxP
Mammalian Cells
1) Any DNA will be incorporated into the host genome: HETEROLOGOUS RECOMBINATION=NO HOMOLOGY REQUIRED. Frequency is about 0.1-1 in 1000 for most cell types. In 1 cell mouse embryos the rate is 1 in 5 when DNA delivered by microinjection.
2) Foreign DNA is incorporated in host chromosomes in a RANDOM manner. Exception: some viral vectors, if viral proteins are supplied in trans (e.g. Epstein-Barr virus vectors).
3) HOMOLOGOUS RECOMBINATION CAN OCCUR, BUT THE FREQUENCY IS MUCH LOWER (1:1-10 million) . A cell will undergo either HETEROLOGOUS OR HOMOLOGOUS RECOMBINATION, BUT NOT BOTH SIMULTANEOUSLY.
In Conventional Transgenic Mice, Injected DNAis Obtained by Heterologous Recombination
Strategy for Homologous Recombination in Mice
Step 1: Gene is Targeted in EMBRYONIC STEM CELLS
Step 2: Targeted ES Cells Are Injected intoBlastocyst Stage Black 6 Embryos and Produce CHIMERIC MICE
Step 3. Chimeric Mice are backcrossed to Black 6:If Germline is Chimeric, then Brown Mice Arise:50% will Have the targeted allele. Breed Heterozygotes to obtain Homozygote Mutants
CRE/loxP Reaction
Targeting Strategy for Conditional “Floxed” Allele
Conventional Transgeneor CRE knockin allele
CRE/loxP Strategy Can also be used to makemore subtle mutations (e.g., point mutations)
5’ Gene Trap Projects in Mouse
1. Insert gene trap vector by retrovirus infection of DNA transfection
2. Isolate individual clones that are neo positive
3. Sequence insertion site to determine which gene has been trapped
4. Confirm that the insertion inactivates the gene
5. Make mice with the ES cells
RosaGeo
pTIGeo
LTR LTR
Vectors Commonly Used for Gene Trapping in Mouse ES Cells
Retrovirus vector: ES cells are infected with thedefective retrovirus vector
Transfection vector: ES cells are transfected with the vector
http://baygenomics.ucsf.edu/overview/welcome.html
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