and several generations are likely to be required to yield animals possessing just one
site of integration and thus generate a stable transgenic line with consistent expres-
sion. Consequently, this approach has not been widely used in mammals but has
been extensively used in the production of transgenic chickens where good alterna-
tive methods of transgenics are not available. Lentiviral transgenesis has also been
used to generate the first transgenic non-human primates that exhibited germline
transmission (Sasaki et al. 2009 ).
The second technological development was reported by Robertson and collea-
gues in 1986. They showed that mouse totipotent embryonic stem (ES) cells could
be genetically modified and made to contribute to the embryo. This opened up the
possibility of specific genetic modification of ES cells in culture and generation of
mice derived from these specifically modified cells. This gene targeting is currently
undertaken via homologous recombination using a targeting vector isogenic with
the target locus in the ES cells apart from the specific modification being intro-
duced. Thus, this technique overcomes the problems associated with microinjec-
tion; the genetic modification is precisely located and is a single copy. This
technique is particularly well suited to disrupting specific genes (knockout) for
functional studies or for the generation of models of inherited monogenic disease in
man. The disadvantage of the ES approach is that the generation of transgenics
takes longer than by pronuclear microinjection and greater molecular and cell
biology skills are required for successful gene targeting and maintenance of the
totipotency of the ES cells.
However, it has not been possible to extend stem cell technology to many other
species. This problem has been overcome by the development of a third technology,
nuclear cloning. In this technique the maternal DNA is removed from an oocyte
and is replaced by a nucleus taken from a cultured cell. Initially this was performed
with nuclei from early embryos but later studies used cells from adult sources.
These donor cells could be genetically modified in culture, using the homologous
recombination approach noted above, before nuclear transfer in the oocyte and
implantation into the foster mother. This technology was first demonstrated with
non-embryonic cells in the sheep by Campbell and colleagues in 1996 with the
production of the famous sheep, Dolly, using the nuclei from cultured cells. This
was shortly followed by the demonstration of mice derived from the nuclei of
somatic cells (Wakayama et al. 1998 ) and calves also derived from nuclei of
somatic cells (Kato et al. 1998 ; Vignon et al. 1998 ). Over a period of 10 years,
a wide range of mammals were cloned from adult somatic cells: pigs (Polejaeva
et al. 2000 ), goats (Zou et al. 2001 ), cats (Shin et al. 2002 ), horses (Galli et al. 2003 )
and dogs (Lee et al. 2005 ).
However there have been significant problems associated with cloning adult
farm animals (Edwards et al. 2003 ). The major limitation is the extreme ineffi-
ciency of the process in terms of live offspring from the number of embryos
generated. This problem does not vary significantly between species or the somatic
cell type. Cloned embryos and foetuses die at all stages of the pregnancies. In
addition, a high proportion of clones are larger than normal and die soon after birth.
Despite these problems some clones are physiologically normal and can reproduce
Genetically Modified Animals and Pharmacological Research 215