1. Lecture 11. Functional Genomics at the Level of the Whole Organism:
Genomic Approaches to Biology

2. 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

3. Goals of Functional Genomics:
1)DNA
2)RNA
3) Protein
4) Whole organism
5) Society
Lander, E The New Genomics: Global Views of Biology. Science 274:

4. Model Systems are especially important.
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.

5. 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
14,000 genes
35-70,000 genes?

6. 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

7. 6000 GENES

10. Targeted Deletions in Yeast

11. http://www-sequence. stanford
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 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

12. Transposon-Mediated Random Mutation Strategy

13. CRE/LoxP Recombination System

15. Random Mutagenesis Strategy

16. Phenotypic Macroarray Analysis
Measure Growth
of Mutants
in 96 well format
Growth
Conditions

17. Cluster Analysis of the Data
Columns:
Growth Conditions
Rows: Various
Mutants

18. After CRE expression:
study protein localization by
immunohistochemistry

19. 2. Gene Targeting in Mouse:
Deletions and Conditional Deletion using CRE/loxP

20. Mammalian Cells
1) Any DNA will be incorporated into the host genome: HETEROLOGOUS RECOMBINATION=NO HOMOLOGY REQUIRED. Frequency is about in 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.

21. In Conventional Transgenic Mice, Injected DNA
is Obtained by Heterologous Recombination

22. Strategy for Homologous Recombination in Mice
Step 1: Gene is Targeted in EMBRYONIC STEM CELLS

23. Step 2:
Targeted ES Cells Are Injected into
Blastocyst Stage Black 6 Embryos
and Produce CHIMERIC MICE

24. 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

25. CRE/loxP Reaction

26. Targeting Strategy for Conditional “Floxed” Allele
Conventional Transgene
or CRE knockin allele

27. CRE/loxP Strategy Can also be used to make
more subtle mutations (e.g., point mutations)

28. 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

29. 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

30.
Bay Genomic Data Base Statistics