Two powerful transgenic techniquesTwo powerful transgenic techniques

Addition of genes by nuclear injectionAddition of genes by nuclear injection Foreign DNA injected into pronucleus of fertilized eggForeign DNA injected into pronucleus of fertilized egg Place injected one-cell embryo back into oviductPlace injected one-cell embryo back into oviduct 25-50% of time DNA integrates randomly into chromosome25-50% of time DNA integrates randomly into chromosome

Targeted mutagenesisTargeted mutagenesis Embryonic stem cells Embryonic stem cells Integrate engineered gene into germ lineIntegrate engineered gene into germ line Homologous recombination mayHomologous recombination may Knockout constructs- nonfunctional gene exchanged for normal Knockout constructs- nonfunctional gene exchanged for normal

gene by homologous recombinationgene by homologous recombination

How How transgenic transgenic mice are mice are createdcreated

Fig. E.7

Using Using transgenic toolstransgenic tools

Determine gene Determine gene functionfunction

SRYSRY locus locus responsible for responsible for production of production of malenessmaleness

Fig. E.9

Application of transgenic Application of transgenic technologytechnology

Transgenic Transgenic expression of myc expression of myc gene provides gene provides information on role information on role of myc in tumor of myc in tumor formationformation

(a) structure of gene(a) structure of gene (b) Northern blot (b) Northern blot

analysisanalysis

Fig. E.10

Using transgenic technology to Using transgenic technology to characterize regulatory regionscharacterize regulatory regions

DNA construct DNA construct containing mouse containing mouse regulatory region of regulatory region of interest is attached interest is attached to to E. coliE. coli reporter reporter gene.gene.

Function ascertained Function ascertained by by -gal expression -gal expression in transgene fetusin transgene fetus

Fig. E..11

Use of transgene Use of transgene technology to map cis-technology to map cis-acting regulatory acting regulatory region of region of Tcp 10bTcp 10btt gene gene

GFP tagging can be used to follow the GFP tagging can be used to follow the localization of proteinslocalization of proteins

Recombinant gene Recombinant gene encoding a GFP encoding a GFP fusion protein at C fusion protein at C terminusterminus

Mouse with GFP-Mouse with GFP-labeled transgene labeled transgene expressed expressed throughout bodythroughout body

Fig. 19.18 c,d

Transgenic technology to Transgenic technology to identify locus identify locus responsible for mutant responsible for mutant phenotypephenotype

Dominant deletion Dominant deletion mutation at T locus mutation at T locus causes short tail.causes short tail.

Transgene mouse with Transgene mouse with pme75pme75 transgene mated transgene mated to mutantto mutant

Normal phenotype Normal phenotype demonstrates deletion of demonstrates deletion of pme75pme75 is cause of short is cause of short tailtail

Fig. E.13

Knocking out a gene in ES cellsKnocking out a gene in ES cells

Targeted mutagenesis to create a mouse model Targeted mutagenesis to create a mouse model for human diseasefor human disease

(Cont’d next slide)Fig. E.14 a-c

ES culture

Fig. E.14 d,e

(cont’d next slide)

Fig. E.14 f (cont’d next slide)

Inheritance pattern Inheritance pattern

Systems Biology – the global study of multiple components Systems Biology – the global study of multiple components of biological systems and their interactionsof biological systems and their interactions

New approach to studying biological New approach to studying biological systems has made possiblesystems has made possible Sequencing genomesSequencing genomes High-throughput platform developmentHigh-throughput platform development Development of powerful computational toolsDevelopment of powerful computational tools The use of model organismsThe use of model organisms Comparative genomicsComparative genomics

Two color DNA microarraysTwo 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

Identification of protein-protein interactionsIdentification of protein-protein interactionsyeast two-hybrid interactionyeast two-hybrid interaction

Fig. 10.32

Quantification of changes in protein Quantification of changes in protein concentration in different cell or tissue statesconcentration in different cell or tissue states

isotope-affinity tag approachisotope-affinity tag approach

Protein labeling strategyProtein labeling strategy