Target Discovery and Validation Reviews and Protocols

170 Houdebine
integrate into the medaka genome (34). The mechanism of action of I-Sce 1 is not
known. It is postulated that the enzyme remains bound to the DNA fragment,
allowing its protection against exonucleases and its unspecific targeting to host
DNA. This approach proved successful in Xenopus but not in mice so far. To
improve DNA binding to sperm, a group recently used a monoclonal capable of
recognizing a surface antigen present in sperm of a number of vertebrates. This
antibody contains a basic amino acid-rich region in its C-terminal end, allowing
a spontaneous binding of DNA and its transfer to oocytes during in vitro or
in vivo fertilization ([35,36]; Fig. 2). Direct DNA transfection into sperm precursors
in seminiferous tubules can be achieved in mice (37). Alternatively, sperm
precursors may be collected, transfected in vitro, and reimplanted into recipient
testis (38–40). These methods are promising but require additional studies
before being used.
2.1.7. Use of Pluripotent or Somatic Cells
Foreign DNA can be transfected into pluripotent cells or somatic cells further
used to generate embryos harboring the transgene. Pluripotent cells, also
known as embryonic stem (ES) cells, can be introduced in developing embryos
at the morula or the blastocytes stage and give birth to transgenic animals capable
of transmitting their transgenes to progeny. In practice, this end can be
achieved with only two mouse strains from pluripotent cells that can be cultured.
Recent studies showed that ES cells also could be obtained in medaka.
Animal cloning by nuclear transfer was rapidly implemented to generate transgenic
sheep (41) and cows (42). Although laborious, the method is more efficient
than DNA microinjection in ruminant pronuclei, and it has not been
adopted for other animals such as rats, rabbits, and pigs. The use of cloning in
these species is therefore restricted to targeted gene transfer.
2.2. Targeted DNA Integration
For several reasons, it is important to target integration of foreign genes. This
approach may avoid uncontrolled position effect of chromatin on transgene
expression. This approach is also the major method to specifically inhibit the
expression of a cellular gene, also known as gene knockout.
2.2.1. Gene Knockout
In all species, although very weakly in plants, a foreign DNA fragment can
replace a genomic DNA by a homologous recombination mechanism. This
replacement implies that relatively long regions of the endogenous and the foreign
DNA have identical sequences (Fig. 3). Homologous recombination is a
rare event, and it cannot be achieved at a sufficient rate in one-cell embryos
after microinjection of the gene constructs. Gene replacement must therefore be