KeywordsAnimal genomesGene therapyPharmingTransgenic farm animals
Transgenic mice
1 Introduction
The technology that allows the addition to or inactivation of specific genes in an
animal’s genome opens up a huge range of opportunities. Genetic manipulation can
be used to create animal models of the human disease, investigate specific gene
function, improve resistance to disease and treat inherited or spontaneous disease.
Before reviewing the use of transgenic animals, it is important to understand the
basics of the production of genetically modified animals and the potential pitfalls
associated with their analysis.
The first method of producing genetically modified animals was reported in the
mouse in 1980 by Gordon and colleagues. The authors used a small pipette to hold a
single cell fertilised egg and another very fine glass pipette to inject a dilute DNA
solution into one or other of the pronuclei. This technology has subsequently been
used to generate a very wide range of genetically modified mammals, including
mouse, rat, rabbit, pig, sheep, goat and cattle (Hammer et al. 1985 ). The process of
integration of the DNA into the host genome is random, generally occurring at only
one site in each embryo but often with multiple copies being integrated at that site
(Brinster et al. 1985 ). A high copy number is often associated with a reduced level
of expression of the transgene (Garrick et al. 1998 ). The random nature of the
integration means that not all of the transgenic animals will express the transgene as
in a proportion of cases the transgene integrates into heterochromatin and so is
silenced. The transgene may also integrate close to a strong promoter/enhancer or
repressor and this may modify the pattern of expression. Finally, the transgene may
integrate into a critical gene that may be responsible for the phenotypic change in
the transgenic rather than the transgene. Consequently, it is generally regarded as
necessary to produce several microinjection transgenics with the same phenotype to
have confidence that the phenotype is due to the transgene. The transgene cassette
design also plays a significant role in determining the level of expression. The
presence of introns generally improves expression compared to a cDNA (Whitelaw
et al. 1991 ) and some sequences upstream of the promoter, locus control regions,
have been shown to confer site-independent expression (e.g. Talbot et al. 1989 ).
Therefore, careful transgene design can improve the efficiency of this technology.
An alternative to microinjection is to use recombinant lentiviruses to transfer a
transgene into an embryo. This approach was first published for mice by Lois et al.
( 2002 ) and its use in farm animals has been recently reviewed (Whitelaw et al.
2008 ). Although this method of transgenesis is much more efficient than micro-
injection, the lentiviral vectors have a limited packaging capacity accommodating a
maximum 8 kb transgene and integration occurs at multiple sites in the genome.
Subsequent breeding of the transgenic founder will lead to segregation of these sites
214 D.J. Wells