Chapter 1 Gene targeting, principles,and practice in mammalian cells

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Gene targeting
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Gene Targeting strategies
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History

• 19771980 homologous recombination
• 19811985 mammalian cells
• 19861991 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
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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

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• 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 lt yeast
• homologous recombinationltrandom integration

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• 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

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• 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

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• Targeting vector types
• - replacement vector most widely used
• - insertion vector

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Replacement vectors

• 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

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• 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

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• 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

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• 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 gt1.5 kb)
• - left homologous arm(5 kb)-positive selection
  marker-right homologous arm(0.5-2kb)

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• Guide lines for the construction of a replacement
  vector

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• 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

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• 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

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• 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

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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

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• 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

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• Guidelines for construction of insertion vectors

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• 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

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• 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

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Maximizing the targeting frequency and selection
of targeted clones

• Random integration predominates
• ---gt design vector to increase the targeted
  integration and select targeted clones
• Insertion vector or replacement vector?
• Length and polymorphism of homologous sequences?
• Selection marker?

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• 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.

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• 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.

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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 ---gt lower the recombination
---gt 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
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• 4.2 Enrichment schems for targeted clones in
  culture
• - transfection of target vector into cells
• ---gt 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

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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
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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
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- 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).
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- 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
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- 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
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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

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• 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-phosphorib
  osylpyrophosphate 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

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• 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

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• 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

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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

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• 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.

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• 6.2 Non-selectable mutations generated by
  co-electroporation
• - 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

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-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..
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• 6.3 Subtle mutations generated with a
  hit-and-run vector
• - utilizes two steps of homologous
  recombination(Fig. 6)
• - insertion vector with both positive and
  negative selection marker outside of homology

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-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
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• 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

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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)

- Loss of an endogeneous gene function -Monitoring
the spatial and temporal expression of an
endogeneous gene -Monitoring the function of a
homologue gene

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• 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