O o d h e a d p u b L i s h I n g L i m I t e d

1.2
The techniques of genetic manipulation
for animals
Genetic manipulation techniques enable the insertion of single genes as
DNA into the genomes of farm animals and to obtain thereby new phenotypes
with changes in production characters. A recent excellent review (Wheeler,
2007) covers the general field of production of transgenic livestock achieved
to the present time. In the case of sheep, single gene variations can be
targeted for wool growth and properties. There are two major strategies: one
is to increase a particular character, a ‘gain of function’, and the other is to
decrease or totally obliterate a production character or ‘loss of function’.
There are several methods for achieving transgenesis, the transference of
selected genes into the genome. The major method used is microinjection of
DNA containing the gene of interest into the pronucleus of a fertilised ovum.
The injected one-cell embryo is then transferred to a recipient female (Wheeler,
2007). A mouse was the first transgenic animal to be produced by microinjection
of the gene for human growth hormone, in the 1980s (Brinster et al., 1981).
The result was an increased growth rate and a larger mouse. Since that time
there have been extensive studies of transgenesis for animal production
including many novel ideas as yet not investigated. Other methods that avoid
microinjection for inserting genes into fertilised ova have been established
including sperm-mediated DNA in which the DNA is adsorbed onto the
sperm surface and transferred into the egg by fertilisation in vitro followed
by implantation into a recipient female (Lavitrano et al., 2006). Similar
procedures include liposome-mediated transfer into the ovum or electroporation
of the transgene DNA into sperm or egg (Ogura, 2002). Retroviruses can be
used as efficient carriers for gene transfer into ova. A development with
potential for transgenic animal production is retroviral gene transduction
into spermatogonia (the precursor cells of spermatozoa) stem cells and then
transplantation to the animal’s testes (Nagano et al., 2001). Transgenic progeny
have been successfully produced in mice by this method but application by
whatever retroviral route in sheep is problematic in the present climate of
opinion on acceptability of transgenic techniques in sheep production.
The ultimate in gene transfer is probably nuclear transfer in which the
nucleus of a somatic or a stem cell is transferred into an enucleated ovum
by microinjection or by cell–cell fusion. The result is a clone of the animal
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Improvement of wool production through genetic manipulation
5
that donated the somatic or the stem cell nucleus. The technique of
therapeutic cloning not only could lead to insertion of a nucleus from cells
of a sheep carrying a gene or genes for a desirable phenotype but could be
a nucleus from a cultured somatic or stem cell after insertion of a specific
gene in a recombination process called ‘knock in’. Alternatively, a gene can
be removed by recombination, so-called gene ‘knock out’. Such procedures
in sheep would be expensive and difficult and have not been investigated,
although a successful specific cloning would not then necessitate frequent
repetition.
All transgenic procedures require the selected gene to be linked to DNA
sequences that can control expression, in particular a promoter, an essential
DNA sequence that can direct expression of the gene in an appropriate tissue
or tissues. The examples for influencing wool growth to be discussed involve
genetic manipulations that could enable genes to function systemically (in
all or most tissues) including the wool follicle or to have expression of new
or modified genes targeted directly to one or more of the seven cellular
layers of the wool follicle (
Fig. 1.1).
In order to affect wool growth and wool properties by inserting novel
genes and directing expression to the wool follicle, a promoter is needed that
is follicle-specific, and one that can even target expression to particular cell
lineage of the follicle. Six different cell layers that are formed in the follicle
from different cell lineages, namely the outer root sheath, three layers of
inner root sheath (IRS), the fibre cuticle and the fibre cortex, differentiate
from the follicle bulb (
Fig. 1.2).
 Each of the cell lineages expresses specific
genes that are potential sources of gene promoters for targeting the different
cell layers of the follicle.