Download Lec206

Next lecture:techniques 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)
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
Mouse ENU mutagenesis
Recent applications of this technology can be seen in:
Nature Genetics,(Aug 2000)Volume 25 pp.440-443 and 444-447
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.
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
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
10 kb
2 kb
X N
B
B
E
B
X
10 kb
2 kb
X N
5’ arm
B
B
E
3’ arm
B
X
10 kb
2 kb
X N
B
5’ arm
ABC
B
E
B
3’ arm
loxP neoR loxP
X
DEF
TKS
X
10 kb
2 kb
X N
B
B
E
loxP neoR loxP
5’ arm
X
B
TKS
3’ arm
Targeting vector
N
X
10 kb
2 kb
X N
B
B
E
B
TKS
5’ arm
X
3’ arm
Targeting vector
N
X
10 kb
2 kb
X N
B
B
E
B
TKS
5’ arm
X
3’ arm
Targeting vector
N
X
10 kb
2 kb
X N
E
X
Homologous recombinant
X
Targeting vector
Randomly integrated
B
X
10 kb
2 kb
X N
E
X
B
X
Homologous recombinant
TKS (gancyclovir)
X
Targeting vector
Randomly integrated
10 kb
2 kb
X N
5’ probe
E
X
Homologous recombinant
B
X
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
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
Cre-mediated deletion
10 kb
2 kb
X N
X
5’ probe
E
B
Homologous recombinant
X
3’ probe
Cre expression vector
X
N
5’ probe
E
Cre-deleted recombinant
B
X
3’ probe
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
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…...
Further references on homologous
recombination in ES cells
• Capecchi, MR (1989) Altering the genome
by homologous recombination. Science.
244:1288-1292.
• Ramirez-Solis, R, Davis, AC and Bradley,
A. (1993) Gene targeting in embryonic stem
cells. Meth. Enzymol. 225:855-875.
Possible phenotypes
• Something related to what you expected
• Something completely unexpected
– No phenotype
– Embryonic lethal
– Complex phenotype-multiple tissues and effects
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
Strategy (from Rajewsky, et. al.)
Transgenic animals (mice)
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
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
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
Model of RNA interference
RNA interference in C. elegans
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