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Lecture 11. Functional Genomics
at the Level of the Whole Organism:
Genomic Approaches to Biology
One goal of Functional Genomics is to
define the function of all genes, and
to define how genes interact to form more
complicated networks responsible for
biological 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)DNA
2)RNA
3) Protein
4) Whole organism
5) 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.
Importance of MODEL SYSTEMS in Genomics
Genome Size and Gene Number in Model Organisms and Man
50 genes
4100 genes
6000 genes
18,000 genes
35-70,000 genes?
14,000 genes
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
http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html
Functional Profiling of the
Saccharomyces 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 SookhaiMahadeo, 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
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 Mutants
in 96 well format
Growth
Conditions
Cluster Analysis of the Data
Columns:
Growth Conditions
Rows: Various
Mutants
After CRE expression:
study protein localization by
immunohistochemistry
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 DNA
is 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 into
Blastocyst 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 Transgene
or CRE knockin allele
CRE/loxP Strategy Can also be used to make
more 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
Vectors Commonly Used for
Gene Trapping in Mouse ES Cells
RosabGeo
LTR
LTR
Retrovirus vector: ES cells are infected with the
defective retrovirus vector
pTIbGeo
Transfection vector: ES cells are
transfected with the vector
http://baygenomics.ucsf.edu/overview/welcome.html
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