Biomedical Engineering Reference
In-Depth Information
regulatory elements for reliable reproduction of endogenous gene expression - sometimes
located at a great distance from the coding sequence. Bacterial artificial chromosome (BAC)
transgenic technology is increasingly used to overcome these difficulties [1]. However, trans-
gene insertion may produce unwanted effects on expression of a nearby, endogenous gene.
Transgenic methods have been described in detail elsewhere [2, 3].
Loss of gene function in mouse can be achieved through gene trap methods [4, 5]. Gene
trap utilizes a reporter gene that is designed to insert randomly into the genome, potentially
interrupting expression of a gene at or near the insertion site. The mutated gene and its
expression patterns can then be identified by means of the inserted reporter. Gene trap can
generate many lines of mice with different disrupted genes; these can be screened based
on a particular phenotype or expression pattern of interest. Recently, the DNA transposons
piggyBAC
and
Sleeping Beauty
have been used to inactivate murine genes by random
insertional mutagenesis [6-8]. Gene trap can be achieved in this manner by incorporating a
reporter gene into the transposon. One advantage of gene trap and transposon-based methods
is that they do not require prior knowledge of the affected gene's sequence or structure.
While gene trap facilitates serendipitous insights into gene function, mutation events may
occur at low frequency, and the investigator has no control over the locus of the mutation.
Gene targeting overcomes many of the limitations of other genetic engineering methods,
as mutations are targeted to inactivate or modify a specific, endogenous gene of interest.
This precise control stems from the use of homologous recombination at the integration
site, combined with negative selection against randomly integrated vector copies. Typically,
targeted mutations have been designed to eliminate gene function, resulting in 'knockout'
mice. However, more elegant strategies are available for gene overexpression, mutation
and deletion, with increasing degrees of temporal and spatial control. These have been
facilitated by the availability of the human and mouse genomic sequences. Gene targeting
is not restricted to alteration of coding regions; gene regulatory regions, untranslated mRNA
regions and noncoding RNAs such as microRNAs can all be specifically targeted in order
to investigate their function.
Owing to the diverse applications of genetic engineering and in the interests of space, this
chapter focuses on selected gene targeting strategies and methods. The protocols included
aim to provide a brief guide to targeting vector design, homologous recombination in mouse
embryonic stem (ES) cells and transfer of mutations to the mouse germline. More in-depth
consideration of the theory and methodology of genetic engineering and gene targeting in
mice can be found in Nagy [9] and Tymms [10].
10.2 Methods and approaches
10.2.1 Principles of targeted gene deletion in mice
Gene targeting is defined as the introduction of site-specific modifications into the genome by
homologous recombination. This powerful technique can be applied to almost any biological
question or phenomenon. Indeed, the
in vivo
functions of over 10 000 genes have now been
analyzed by gene targeting, and over 500 different mouse models of human disorders have
been created. The technique of gene targeting in mice was developed as a combination of
two major breakthroughs, recognized by the 2007 Nobel Prize in Physiology or Medicine,
awarded to Mario Capecchi, Martin Evans and Oliver Smithies. The first advance was the
ability to culture mouse ES cells that have the potential to contribute to the germline of
