1. Two powerful transgenic techniques Addition of genes by nuclear injection Addition of genes by nuclear injection Foreign DNA injected into pronucleus of fertilized egg Foreign DNA injected into pronucleus of fertilized egg Place injected one-cell embryo back into oviduct Place injected one-cell embryo back into oviduct 25-50% of time DNA integrates randomly into chromosome 25-50% of time DNA integrates randomly into chromosome Targeted mutagenesis Targeted mutagenesis Embryonic stem cells Embryonic stem cells Integrate engineered gene into germ line Integrate engineered gene into germ line Homologous recombination may Homologous recombination may Knockout constructs- nonfunctional gene exchanged for normal gene by homologous recombination Knockout constructs- nonfunctional gene exchanged for normal gene by homologous recombination

2. How transgenic mice are created Fig. E.7

3. Using transgenic tools Determine gene function Determine gene function SRY locus responsible for production of maleness SRY locus responsible for production of maleness Fig. E.9

4. Application of transgenic technology Transgenic expression of myc gene provides information on role of myc in tumor formation Transgenic expression of myc gene provides information on role of myc in tumor formation (a) structure of gene (a) structure of gene (b) Northern blot analysis (b) Northern blot analysis Fig. E.10

5. Using transgenic technology to characterize regulatory regions DNA construct containing mouse regulatory region of interest is attached to E. coli reporter gene. DNA construct containing mouse regulatory region of interest is attached to E. coli reporter gene. Function ascertained by  -gal expression in transgene fetus Function ascertained by  -gal expression in transgene fetus Fig. E..11

6. Use of transgene technology to map cis- acting regulatory region of Tcp 10b t gene Use of transgene technology to map cis- acting regulatory region of Tcp 10b t gene

7. GFP tagging can be used to follow the localization of proteins Recombinant gene encoding a GFP fusion protein at C terminus Recombinant gene encoding a GFP fusion protein at C terminus Mouse with GFP- labeled transgene expressed throughout body Mouse with GFP- labeled transgene expressed throughout body Fig. 19.18 c,d

8. Transgenic technology to identify locus responsible for mutant phenotype Transgenic technology to identify locus responsible for mutant phenotype Dominant deletion mutation at T locus causes short tail. Dominant deletion mutation at T locus causes short tail. Transgene mouse with pme75 transgene mated to mutant Transgene mouse with pme75 transgene mated to mutant Normal phenotype demonstrates deletion of pme75 is cause of short tail Normal phenotype demonstrates deletion of pme75 is cause of short tail Fig. E.13

9. Knocking out a gene in ES cells

10. Targeted mutagenesis to create a mouse model for human disease (Cont’d next slide)Fig. E.14 a-c

11. ES culture Fig. E.14 d,e (cont’d next slide)

12. Fig. E.14 f (cont’d next slide)

13. Inheritance pattern

14. Systems Biology – the global study of multiple components of biological systems and their interactions New approach to studying biological systems has made possible New approach to studying biological systems has made possible Sequencing genomes Sequencing genomes High-throughput platform development High-throughput platform development Development of powerful computational tools Development of powerful computational tools The use of model organisms The use of model organisms Comparative genomics Comparative genomics

15. Two color DNA microarrays Two separate cDNA samples, one from normal yeast, and the other from mutant yeast labeled with red and green fluorescent dyes and hybridized to PCR microarray Fig. 10.25

17. Identification of protein-protein interactions yeast two-hybrid interaction Fig. 10.32

18. Quantification of changes in protein concentration in different cell or tissue states isotope-affinity tag approach

19. Protein labeling strategy