Transcript Genetic engineering and microarrays

Two powerful transgenic techniques
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Addition of genes by nuclear injection
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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
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Embryonic stem cells
Integrate engineered gene into germ line
Homologous recombination may
Knockout constructs- nonfunctional gene exchanged for normal
gene by homologous recombination
How
transgenic
mice are
created
Fig. E.7
Using
transgenic tools
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Fig. E.9
Determine gene
function
SRY locus
responsible for
production of
maleness
Application of transgenic
technology
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Fig. E.10
Transgenic
expression of myc
gene provides
information on role
of myc in tumor
formation
(a) structure of gene
(b) Northern blot
analysis
Using transgenic technology to
characterize regulatory regions
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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
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Use of transgene
technology to map cisacting regulatory
region of Tcp 10bt gene
GFP tagging can be used to follow the
localization of proteins
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Fig. 19.18 c,d
Recombinant gene
encoding a GFP
fusion protein at C
terminus
Mouse with GFPlabeled transgene
expressed
throughout body
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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
Knocking out a gene in ES cells
Targeted mutagenesis to create a mouse model
for human disease
Fig. E.14 a-c
(Cont’d next slide)
ES culture
(cont’d next slide)
Fig. E.14 d,e
Fig. E.14 f (cont’d
next slide)
Inheritance pattern
Systems Biology – the global study of multiple components
of biological systems and their interactions
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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
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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
Identification of protein-protein interactions
yeast two-hybrid interaction
Fig. 10.32
Quantification of changes in protein
concentration in different cell or tissue states
isotope-affinity tag approach
Protein labeling strategy