Biomedical Engineering Reference
In-Depth Information
advantage of PCR screening, with one primer derived from the newly introduced neomycin
resistance cassette and the other hybridizing to a genomic sequence outside the SHA. The
SHA length is restricted to 1 kb to allow for efficient amplification by PCR during screening
of ES cell DNA.
The high degree of precision of genetic manipulation through gene targeting in mice
comes at some expense. The procedures can be involved, labor intensive, relatively costly
and lengthy. Automated approaches to gene targeting in ES cells and subsequent production
of gene-targeted mice may be suited to the needs of some laboratories [27, 28].
10.2.2 Strategies for gene targeting in mice
Because of the extensive variety of mutations and sequence manipulations that are possible
through gene targeting, this chapter will discuss a selection of the most common targeting
strategies as listed below. In all cases, a targeting vector must be designed such that chro-
mosomal sequences are replaced by vector sequences through homologous recombination of
the flanking homology regions. A modified pBluescript (pBSK) plasmid may be employed
as a suitable targeting vector (Figure 10.1d).
1
Knockout
: Targeting constructs for generation of null mutants typically consist of
the target gene sequence into which a loss-of-function mutation has been engineered
(Figure 10.1a). This can be achieved by replacing the initiation codon (ATG) and/or
an exon (encoding the functional domain of a protein) with the neomycin resistance
cassette. In order to effectively eliminate gene function, this replacement should also
incorporate a translational termination codon, while generating a frameshift mutation
that precludes alternative splicing. It is important to note that transcription from the
strong promoter driving the neomycin resistance gene may interfere with expression of
neighboring genes, thus confounding phenotypic analysis [29, 30]. Thus, the neomycin
resistance cassette should be flanked by two FLP recombination target (FRT) sites,
allowing for FLP recombinase-mediated excision [31]. This is achieved by crossing
with a mouse expressing FLP recombinase in the germline [32]. Null mutations may
be embryonic lethal, restricting the study of gene function to early development. In
other cases, compensation for constitutive gene deletion may yield phenotypes that
underestimate gene function. These pitfalls can be avoided through conditional or
inducible mutations described below.
2
Knockin
: Knockin mutations are generated through sequence replacement or insertion
at a specific gene locus (Figure 10.1b). Possible applications include the introduction
of specific point mutations or the fusion of reporter genes such as green fluorescent
protein (GFP) or
β
-galactosidase. Such reporter gene fusions facilitate accurate analysis
of endogenous gene expression patterns, while manipulations such as point mutations
allow more subtle dissection of gene function. In addition, overexpression can be achieved
by knockin of the gene of interest at the
ROSA26
locus. This locus is regarded as one
from which proteins can be expressed ubiquitously at a moderate level [33].
3
Conditional deletion
: In order to overcome some potential difficulties associated with
constitutive deletion and to analyze gene function in a particular cell, tissue and/or
developmental stage of interest, gene targeting can be combined with the powerful
Cre/loxP system (reviewed in Refs. [34, 35]). Cre recombinase efficiently catalyzes
