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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