Next%20lecture:techniques%20used%20to%20study%20the%20role%20of%20genes%20in%20develpoment

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Next lecturetechniques used to study the role of
genes in develpoment

• Random genetics followed by screening
• Targeted mutagenesis (gene knockout)
• Transgenic animal models
• Dominant negative mutant molecules
• Antisense RNA interference
• RNA interference (c. elegans-website 4.8)

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Random genetics

• Chemical mutagen/gene disrupting agent
• Screening for a phenotype
• Dominant mutations can be found easily
• Recessive mutations require breeding
• Frequently used with model organisms
• Especially Drosophila (lectures in late Feb.)
• Becoming newly popular in the mouse

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Mouse ENU mutagenesis
Recent applications of this technology can be
seen in Nature Genetics,(Aug 2000)Volume 25
pp.440-443 and 444-447
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Trans-heterozygous phenotypes

• When two genes are in the same pathway mutants
  heterozygous for both genes will display a
  phenotype even though each individual
  heterozygous mutant does not
• Can be combined with ENU mutagenesis to screen
  for genes in the same pathway as another known
  knocked out gene.

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Targeted mutagenesis (knockout)

• Determine the action of a known, cloned gene in a
  developmental process
• Removes a segment of the known gene by homologous
  recombination
• Required elements
• Mapped genomic clone for the gene of interest
• Embryonic stem (ES) cells
• A lot of repetitive work

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Points to make

• Genomic clone should come from the same mouse
  strain from which the ES cell is derived (common
  strain 129SV)
• Making of targeting construct. 6kb of homologous
  arms with appropriately arranged selectable
  markers for positive and negative selection
• A screening strategy involving two probes

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10 kb
2 kb
X N B B E
B X
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10 kb
2 kb
X N B B E
B X
5 arm
3 arm
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10 kb
2 kb
X N B B E
B X
5 arm
3 arm
loxP neoR loxP
TKS
ABC
DEF
X
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10 kb
2 kb
X N B B E
B X
loxP neoR loxP
TKS
5 arm
3 arm
X
N
Targeting vector
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10 kb
2 kb
X N B B E
B X
TKS
N
5 arm
3 arm
X
Targeting vector
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10 kb
2 kb
X N B B E
B X
TKS
N
5 arm
3 arm
X
Targeting vector
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10 kb
2 kb
X N E
B X
X
Homologous recombinant
X
Targeting vector Randomly integrated
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10 kb
2 kb
X N E
B X
X
Homologous recombinant
TKS (gancyclovir)
X
Targeting vector Randomly integrated
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10 kb
2 kb
X N E
B X
X
Homologous recombinant
5 probe
3 probe
X
Targeting vector Randomly integrated
Note, probes are OUTSIDE the homology arms.
Therefore, they will only detect the endogenous
locus and the recombinant
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Removing the selection marker

• Cre recombinase deletes sequences between two
  lox-P sites in the same orientation
• Transiently transfect a vector expressing the cre
  recombinase for deletion
• Grow clones again and screen for the deletion by
  southern blot as before
• Especially necessary in studying knockouts of
  genetic regulatory sequences

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Cre-mediated deletion
10 kb
2 kb
X N E
B X
X
Homologous recombinant
5 probe
3 probe
Cre expression vector
X N E
B X
5 probe
3 probe
Cre-deleted recombinant
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The next steps

• Grow up clones with correct recombinant
• Inject ES cells into mouse blastocyst (d3.5)
• The ES cells will integrate themselves into the
  blastocyst and mouse will be a chimera
• Usually the ES cell strain has a different coat
  color than the blastocyst strain so that the
  marbleized mice can be easily seen

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And then.See also fig 4.19 on page 98 of Gilbert

• Breed the chimeric mice to normal mice
• If the ES cells contributed to the germline the
  babies which contain the mutation will have the
  coat color of the ES cell
• These mice are only HETEROZYGOUS for the targeted
  gene
• These mice need to be bred to homozygous
• Analyze the phenotype...

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Further references on homologous recombination in
ES cells

• Capecchi, MR (1989) Altering the genome by
  homologous recombination. Science. 2441288-1292.
• Ramirez-Solis, R, Davis, AC and Bradley, A.
  (1993) Gene targeting in embryonic stem cells.
  Meth. Enzymol. 225855-875.

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Possible phenotypes

• Something related to what you expected
• Something completely unexpected
• No phenotype
• Embryonic lethal
• Complex phenotype-multiple tissues and effects

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Conditional gene targeting

• Tissue-specific knockout of a gene
• Avoids embryo lethality
• Avoids complex phenotypes
• Inducible knockout
• Allows before and after type analysis
• Model of acquired mutation rather than inherited
  mutation

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Strategy (from Rajewsky, et. al.)
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Transgenic animals (mice)
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Points to make

• Transgene should be free of vector DNA
• Transgene must be rigorously purified
• Transgene integration is a bit inefficient
• Founder mice are sometimes mosaic
• Need to outcross the mice to wild type mice
• The transgene is not always expressed
• Position-effect-variegation

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Transgenesis and developmental studies

• Inappropriate or overexpression of a gene
• Dominant negative mutant gene expression
• Reporter gene expression for lineage tracing
• Fluorescent proteins (GFP, YFP)
• Beta-galactosidase (X-gal staining)
• Transcriptional regulatory elements
• Assuming a position-independent system

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Antisense and RNA interference

• Overexpression of anti-sense RNA
• Not the method of choice though it has worked in
  some instances.
• RNA interference in c. elegans
• Website 4.8

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Model of RNA interference
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RNA interference in C. elegans
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Next lectures Differential Gene expression

• Chapter 5 and websites on syllabus
• Epigenetic control mechanisms
• Histone modification
• DNA methylation
• Nucleosome disruption machines
• Promoters and enhancers
• Old and new models of enhancer function
• Novel transcriptional control sequences