Two powerful transgenic techniques

1
Two powerful transgenic techniques

• Addition of genes by nuclear injection
• Foreign DNA injected into pronucleus of
  fertilized egg
• Place injected one-cell embryo back into oviduct
• 25-50 of time DNA integrates randomly into
  chromosome
• Targeted mutagenesis
• Embryonic stem cells
• Integrate engineered gene into germ line
• Homologous recombination may
• 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
• 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
• (a) structure of gene
• (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.
• Function ascertained by b-gal expression in
  transgene fetus

Fig. E..11
6

• Use of transgene technology to map cis-acting
  regulatory region of Tcp 10bt gene

7
GFP tagging can be used to follow the
localization of proteins

• Recombinant gene encoding a GFP fusion protein at
  C terminus
• Mouse with GFP-labeled transgene expressed
  throughout body

Fig. 19.18 c,d
8

• Transgenic technology to identify locus
  responsible for mutant phenotype
• Dominant deletion mutation at T locus causes
  short tail.
• Transgene mouse with pme75 transgene mated to
  mutant
• 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
(Contd next slide)
Fig. E.14 a-c
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ES culture
(contd next slide)
Fig. E.14 d,e
12
Fig. E.14 f (contd 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
• Sequencing genomes
• High-throughput platform development
• Development of powerful computational tools
• The use of model organisms
• 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
16
(No Transcript)
17
Identification of protein-protein
interactionsyeast two-hybrid interaction
Fig. 10.32
18
Quantification of changes in protein
concentration in different cell or tissue
statesisotope-affinity tag approach
19
Protein labeling strategy