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

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. 1996. The New Genomics: Global Views of Biology. Science 274: 536-539.

4. 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 14,000 genes 6000 genes 18,000 genes 4100 genes 50 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. Functional Profiling of theSaccharomyces cerevisiae Genomeclick 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

12. Transposon-Mediated Random Mutation Strategy

13. CRE/LoxP Recombination System

14. http://ygac.med.yale.edu/triples/triples.htm

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 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.

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. http://baygenomics.ucsf.edu/overview/welcome.html Bay Genomic Data Base Statistics