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
- 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
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) `
- 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
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