Transcript Chapter 1 Gene targeting, principles,and practice in

Gene targeting
Gene Targeting strategies
History
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1977–1980: homologous recombination
1981–1985: mammalian cells
1986–1991: embryonic stem cells
1991 to present: The use of gene targeting
to evaluate the function of gene
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Gene targeting, principles,and
practice in mammalian cells
Introduction
• An introduced gene fragment recombines
with the homologous sequence in the
genome(homologous recombination) gene targeting
• A modified gene fragment can replace the
endogenous wild type gene-phenotypic
alteration can be assessed in the
organism
• Examples of gene targeting
- gene targeting a fibroblast cell line with a
selectable artificial locus
- beta-globin gene in erythroleukaemia cells
• Efficiency of gene targeting in mammalian
cells is low
mammalian cell < yeast
homologous recombination<random
integration
• Targeting vectors
- must have a (modified) sequence
homologous with target gene
- selection marker: to select transfected
cells and increase the targeted
recombination products
- positive selection marker
- negative selection marker
• Positive selection marker
- isolate rare stably transfected cells
- inserted within the homologous gene in
the vector to make it non-functional and
used as mutagen.
• Negative selection marker
- eliminates random insertions and insertion
of heterologous components
• Targeting vector types
- replacement vector: most widely used
- insertion vector
Replacement vectors
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Homologous sequence
Positive selection marker
Negative selection marker(optional)
Double homologous recombination should
occur flanking vector
components(heterologous sequences) are
eliminated(excised)
• Linearization site is outside of homologous
region
• Design considerations of a replacement
vector
- general mutation method: insertion of
positive selection marker in the exon or
replacing part of exon by the positive
selection marker-must confirm the
targeted gene is null by RNA or protein
analysis since truncated form of pretein
may retain some activity
- mutated exon may not be recognized by
splicing machinery and skipped -this
deleted the mutated exon in RNA
• Avoid in-frame deletion because it may
produce functional protein
• Large deletion is recommended
- targeting frequency
19 kb deletion = small deletion
• Too mucjh deletion may affect multiple
genes
• Length of homologous sequences should
be 5-8 Kb
• Screening of targeted cells
- position of selection markers with respect
to homologous sequences
- PCR or southern blot analysis
- homologous sequence shoud be longer
than 500 bp( usually >1.5 kb)
- left homologous arm(5 kb)-positive
selection marker-right homologous
arm(0.5-2kb)
• Guide lines for the construction of a
replacement vector
• Recombinant alleles generated by
replacement vectors
- vector concatemers, circles produce
undesirable products-entire vector
insertion
- the entire vector insertion can be
eliminated by negative selection and PCR
• Replacement vector: screening for
targeted events
PCR
primer position: one must be from positive
selection marker gene, the other must be
from outside of cloned homologous
sequence in the vector
• Southern blot anaylsis
- probe position : must be from outside of
the cloned homologous regions
- a restriction site should exist just outside
of the probe region
Insertion vectors
• Linearization site is in the homologous
sequence
• Inserted into the target site by single
reciprocal recombination
• 5-20 fold higher frequency than
replacement vectors
• Entire vector sequences is integrated:
duplication of homologous region
separated by heterologous sequences
• Vector design for insertion vectors
- a homology region with a unique
linearization site
- a positive selection marker: within the
homology region or plasmid
backbone(preferred)
-bacterial plasmid backbone
• Guidelines for construction of insertion
vectors
• Screening for recombinant alleles
generated with insertion vectors
PCR : include a primer from gap repair
region and the other primer from
heterologous vector(gap : 1-4 kb)
* gap: deleted 1-4 kb homologous region
by restriction digestion and religate and
trasnsform into E. coli. If no suitable
restriction sites are unavailble, use small
linker DNA with a unique restriction site
• Southern blot
- probe region from outside of homologous
region
- or gap probe
Test genomic DNA digestion: use restriction
sites that do not cut within vector
Maximizing the targeting frequency and
selection of targeted clones
• Random integration predominates
---> design vector to increase the targeted
integration and select targeted clones
Insertion vector or replacement vector?
Length and polymorphism of homologous
sequences?
Selection marker?
• Homology to the target locus:
– length of homology
-The longer the homology, the higher
frequency
- Ideal length 5-10 kb
- In replacement vector, positive selection
marker devides the homology asymmetrically
into long arm and short arm and short arm
should be 2 kb or longer but PCR amplifiable.
• In insertion vector, the double strand break
should be at least 1.5 kb away from the
large selection marker. If subtle mutations
are made in the homology the location of
the double strand break does not greatly
affect the frequency.
4.1.2 Degree of homology
- sequence variation between two homologous
elements can affect recombination frequency
- DNA mismatch repair is involved in repairing the
mismatches and heterologies
---> lower the recombination
---> recombination of non-isogenic vectors are
elevated to the levels of isogenic vectors in
mismatch repair mutant cells
- DNA used to construct the targeting vector
should be isogenic to the cells used in the
targeting experiments
• 4.2 Enrichment schems for targeted clones in
culture
- transfection of target vector into cells
---> integrate into the target site or random sites
- factors affecting targeting: location of the target
site, length of homology, vector type(insertion or
replacement)
- negative selection marker, trapping of promoter
or poly(A) site of the endogenous gene
4.2.1 Positive-negative selection for targeted
clones
- selection against random integration
- applicable to replacement vectors(Fig 5A, 5B)
- positive selection : select for all types of
integration
- negative selection : select against random
integration by killing the clones
- enrichment by negative selection : 2- 20 fold
4.2.2 Positive selection for targeted clones:
promoter, enhancer, and polyadenylation
trap targeting vectors
- use the transcriptional activity of the
endogenous target gene to express the
positive coding region cloned within the
exon of targeting vector.
- the target gene must be transcriptionally
active in the cells
- promoter trap vector: positive selection
cassette is cloned in-frame with the
endogenous translated product, or if the
positive selection cassette has its own
initiation codon, it can be placed upstream
or in place of the nominal translational
initiation site (Fig. 5E, 5F, efficiency: 100
fold enrichement, works for both
replacement vector and insertion vector).
- enhancer trap vector:
*similar to promoter trap vector.
*use a weak position dependent
promoter.
*vector designing is simple because a
fusion transcript/gene product is not
required
- polyadenylation trap vector
* trap polyadenylation signal to generate
stable transcript
* positive selection cassette has its own
promoter
* applicable to insertion vector and
replacement vector
* 5-50 fold enrichment
5. Selection markers
• Positive selection marker is necessary to
isolate stably transfected cells
• Negative selection marker is to eliminate
random integration
• Marker type: domonant marker(eg.
Neomycine gene), recessive marker( eg.
Hprt gene)
• Selection markers: Table 1
• 6TG is first converted to 6TGMP by Hprt in the purine
salvage pathway (fig. 1, (Calabresi and Parks, 1985 )).
The biological activity of this product is several-fold.
First, 6TGMP works as a pseudofeedback inhibitor of
glutamine-5-phosphoribosylpyrophosphate
amidotransferase and blocks purine biosynthesis.
Second, 6TGMP inhibits IMP dehydrogenase and thus
purine interconversion. The net consequence of this
activity is a block of the synthesis and utilization of
purine nucleotides
• FIAU is converted to toxic compound by TK
• 5.1 promoters and polyadenylation sites
used for selection markers
- Positive selection marker: position
independent promoters: PGK, RNAPII
- Negative selection marker: MC1 promoter
- RNA processing signal: polyadenylation
signal, terminator
• 5.2 Effects of selection markers on
phenotypes
- marker gene may affect other gene
expression.
- may remove marker gene after targeting
to avoid undesirable effects
- marker gene removal can be readily
accomplished by Cre-loxP system
6. Generating subtle mutations with
gene targeting techniques
• Sometimes subtle changes in nucleotide
level in both coding region or control
region are improtant in full understanding
of gene function
• 4 techniques are available to introduce
small mutations
• 6.1 Subtle mutations generated by
microinjection
- 20% of the microinjected cells integrate
the injected DNA
- each clone should be expanded and
tested for gene replacement by southern
blot analysis
- not widely used: successfully used for
fibroblast and ES cells but have not been
repeated.
• 6.2 Non-selectable mutations generated by coelectroporation
- co-introduction of a positive selectable marker
and a non-selectable vector
- co-introduction will result in 3 categories of
clones : non-targeted clones, clones with
integratged concatemers of targeting vector and
the selection marker in the target site, and
clones targeted by simple homologous
recombination in which selection marker has
integrated in another locus
-to screen the true recombinant by PCR
and/or southern blot analysis design
unique PCR primers or southern probes
by changing wobble bases or generate a
novel restriction site.
- exclude integration of concatemers of the
selection cassette and vector in the target
site by digesting genomic DNA with a
restriction enzyme that does not cut within
the plasmids..
• 6.3 Subtle mutations generated with a hitand-run vector
- utilizes two steps of homologous
recombination(Fig. 6)
- insertion vector with both positive and
negative selection marker outside of
homology
-1st step
*homologous recombination and positive selection are
used to generate a duplication at target locus
-2nd step
*spotaneous intrachromosomal recombination(pop-out)
between the duplicated homologous region
* a unique I-Scel endonuclease site can be included in
the vector to increase pop-out
* negative selection
* uneven sister chromatid exchange is more frequent
• 6.4 Subtle mutations generated by double
replacement
- two round of homologous recombination
Fig. 7
- replacement vectors
- 1st step: replacement vector with positive
and negative marker in the homology
- 2nd step : replacement vector without
any selectable marker but with a mutated
homologous sequence-negative selection
7. Knock-in targeting vectors: simultaneous
study of gene function and expression
• Replacing an endogenous gene with
another gene (a homologue gene, a
marker gene or a reporter gene under the
transcriptional control of an endogenous
gene)
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- Loss of an endogeneous gene function
-Monitoring the spatial and temporal expression
of an endogeneous gene
-Monitoring the function of a homologue gene
• No endogenous sequences(regulatory
sequences) should be deleted after
targeting
• Positive marker should be deleted after
targeting by use of Cre-loxP system
• Knock-in strategy : Fig. 8
• Use of IRES : Fig. 9