Target Discovery and Validation Reviews and Protocols

222 Vijayaraj, Söhl, and Magin
1.2. Methodological Considerations
1.2.1. Design of Targeting Vectors
Targeted gene manipulation can be accomplished by the use of either
replacement or insertion vectors (103). Replacement vectors are used to disrupt
target gene function by deleting specific gene sequences and by replacing them
with heterologous DNA, usually a drug selection gene or a marker gene to
analyze target gene expression. Insertion vectors are used to disrupt gene function
by inserting heterologous DNA, but they also allow for the introduction of
more subtle genetic alterations such as point mutations. Here, we focus first
on replacement vectors and describe approaches to modify a gene or gene
fragment constitutively or conditionally. Then, we describe how large-scale
genomic deletions are carried out using insertion vectors. All gene-targeting
vectors should be constructed from isogenic DNA (i.e., from the same mouse
strain as the embryonic stem [ES] cell line to be used). Deviation from this rule
will decrease targeting frequencies by 10- to 1000-fold.
1.2.2. Constitutive and Conditional Alterations
Most IF genes have a gene structure well suited for gene targeting, because
they span approx 10 kb of genomic DNA. To generate a null allele or to introduce
consecutively several independent mutations, it is desirable to delete the
coding sequences completely from the genome. As long as no more than 20 kb
of genomic DNA is replaced, the efficiency of gene targeting remains high.
A functional null allele also can be generated by deleting the core promoter and
the first coding exon. In such a case, alternative start codons should be considered
that can be removed by site-directed mutagenesis. Figure 4A illustrates
typical replacement-type targeting vectors carrying two segments of sequences
homologous to the gene of interest. A short arm of ~1.5 kb to allow for easy
PCR detection and a long arm of 4–6 kb is required. For PCR detection, one
PCR primer resides in the selectable marker gene, and the other primer resides
outside the region of homology. Homologous arms can either be isolated from
genomic libraries, or, more conveniently, by using long-range PCR from
genomic DNA preparations or bacterial artificial chromosome clones carrying
the gene of interest. Vector arms flank the part of the gene to be manipulated
and carry between them a positive selectable marker gene. Upon homologous
recombination between vector arms and the corresponding genomic DNA, the
selectable marker gene replaces the genomic sequences and creates a modified
gene. In our hands, this approach has resulted in targeting frequencies between
1:5 and 1:35 positive ES cell clones (21,34,75,81,104). The selectable marker
genes that code for resistance against neomycin, hygromycin, puromycin, or
hypoxanthine phosphoribosyl transferase (HPRT) are most commonly used. In