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

partially degrades the exocuticle and A-layer (
Fig. 1.5)
 of the cuticle cells,
resulting in a marked lowering of the differential friction. Binding a polymer
to the outside surface of the fibre increases this effect.
1.5.2
The expression of keratin and novel proteins in
the fibre by transgenesis
Any genetic manipulation of the fibre contemplated to alter the physical
properties in some way, such as increasing fibre strength, has to be based on
1.6
 The cuticle of a wool fibre showing the overlapping flattened
cells and their edges in profile.
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Advances in wool technology
14
the known structure of cortex and cuticle as outlined here. The protein
composition of the cortex or the cuticle could be modified by either increasing
or decreasing the abundance of one of the proteins. According to genomic
studies of human hair there are 10 functional genes for Type I and 10 for
Type II keratin IFs (Langbein and Schweizer, 2005) and for the matrix
proteins there are 17 glycine/tyrosine KAP genes and about 67 genes for the
sulphur-rich KAPs (Rogers et al., 2006). All of the genes are present in the
genome as large domains in several chromosomes and apparently evolved
through gene duplication and mutation. Their numbers and domain organisation
could be expected to be similar in sheep according to protein analyses and
current knowledge from gene characterisation. The multiplicity of members
in these protein families and their similarity of sequence suggest that the
molecular structure of the filament and matrix can tolerate variations in the
relative abundance of the different chains. Indeed, wools from different
sheep breeds maintain the basic structure of filament and matrix despite
variation in protein composition. Hence it can be concluded that transgenic
manipulation is a valid approach to obtaining changes in fibre composition
by over-expression or under-expression of selected keratin genes.
At present, only over-expression of a limited number of single genes in
the wool cortex have been experimentally tested in transgenic sheep. The
timing of gene expression in the cortex depends on the promoter that is
linked to the gene to be expressed. The choice of promoters from KIF and
KAP genes, in principle, could be utilised to allow a transgene to be expressed
at stages of wool fibre formation that are either early, midway or late and
would be expected to have differing effects on fibre properties. An important
aspect to be noted is that experiments conducted so far have indicated that
promoters of the KIF genes are more active than promoters of the KAP
genes. It is known that the global control of gene expression occurs through
certain loci in genomes. Hair and wool keratin genes exist in domains in the
chromosomes, and characterisation of such regions could be important for
the fine-tuning of transgenic gene expression and thus for the modulation of
consequent changes to wool structure and growth.
The first objective in the transgenesis investigations supported by the
Australian Cooperative Research Centre (CRC) for Premium Quality Wool
and the agencies supporting the CRC was to devise strategies that might
improve the strength and/or elasticity of the fibre. In the initial experiments,
a sheep gene for a Type II KIF called K2.10 was chosen to provide the
promoter for expressing transgenes targeted to the wool follicle cortex that
activates expression relatively early in cortical cell differentiation. This gene
promoter had already been shown to be functional in mouse hair follicles
(Powell and Rogers, 1990) and in merino wool follicles (Bawden
et al., 1998). A promoter–gene construct was made, comprising the K2.10
promoter linked to other regulatory sequences with a cloning site to enable
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Improvement of wool production through genetic manipulation
15
insertion of chosen DNA sequences adjacent to, and downstream from, the
promoter. In order to reduce interference to gene expression by host
chromosomal DNA, a functional insulator sequence was also included in the
basic construct (Bawden et al., 1998, 2000).
The experiments to enhance intrinsic strength of wool fibres involved
introducing more cysteine-rich KAP proteins into the cortical cell structure
on the basis that they could increase the level of crosslinking of disulphide
bonds. An alternative choice was to introduce another crosslink, the isopeptide
crosslink, by expressing the transglutaminase enzyme responsible for catalysing
the formation of these links between lysine and glutamine side-chains in
proteins. The other aspect considered for change was fibre elasticity and the
route chosen for investigation was to attempt to convert the cortex to a
predominantly orthocortical (‘soft’) type by increasing the level of one of
the glycine/tyrosine-rich proteins or by increasing the proportion of KIFs in
the cortex by co-expression of Type I and Type II wool KIF transgenes.
These manipulations were shown by in situ hybridisation to achieve the
expected expression of the proteins in the wool cortex. Furthermore, the
presence of the expressed proteins could be visualised by electron microscopy
except in the case of co-expression of the Type I and Type II genes. These
transgenic experiments demonstrated that the phenotypes could be inherited
by following generations. The load-bearing capacity and the extensibility of
fibres from the transgenic animals were evaluated and in all cases (except for
the Type I and Type II wool KIF co-expression) the overall result was that
these physical properties were reduced in comparison with fibres of age-
matched non-transgenic siblings. However, preliminary tests on several
transgenic wools woven into fabric indicated that there was a significant
increase in comfort (M. Huson, personal communication). It is possible that
the prickle property was reduced by increasing the flexibility of the fibres.
The expression data obtained so far indicates that the level of transgene
expression is such that even if the transgene product inserts into the native
KIF-matrix structures excess proteins are also ‘dumped’ in the cells as
amorphous masses. This situation was observed for instances when the matrix
protein KAP6 or a Type I IF protein (without its Type II partner) were over-
expressed 
(Fig. 1.7).
Another consequence of transgene expression that has been observed is
that expression of endogenous genes is reduced, presumably because the
protein synthetic machinery can be overwhelmed by the transgene expression.
Future problems in transgenic research to be solved are to produce transgene
constructs that:
• have promoters that are active at the correct time of cortical differentiation;
• include DNA elements (microRNAs) found in the genome that can control
gene expression. Synthetic anti-sense gene sequences also can be designed
to target specific genes and reduce the level of their expression and
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Advances in wool technology
16
1.7
 (a) Transmission electron micrograph of a wool fibre from a
transgenic sheep expressing a transgene for KAP6 protein directed to
the wool cortex. Normal orthocortex ‘1’ and paracortex ‘2’ that has
an orthocortex appearance resulting from the deposition of KAP6
protein. (b) Transmission electron micrograph of a wool fibre from a
transgenic sheep expressing a transgene for a Type I IF protein
directed to the wool cortex. Deposits of IF protein darker than the
normal keratin are present throughout the cortex.
(a)
(b)
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Improvement of wool production through genetic manipulation
17
constitute part of that approach. It is pertinent to mention that although
fibre stiffness mentioned above is a function of the keratin proteins in
the cortex, the cells of the cortex are enveloped by membranes (m in 
Fig.
1.3)
 that appear to consist of stiff crosslinked proteins, one of them
might be involucrin and another, loricrin. It is possible that any ‘stiffening’
effect could be could be reduced or eliminated using anti-sense technology
on these proteins and thereby increase fibre flexibility.
The modification of wool properties by expressing foreign protein in
wool, especially those that have a fibrous molecular structure, has been
proposed but awaits investigation. Collagen, elastin and silk are candidate
fibrous proteins and if expressed at sufficiently high levels in the cortex they
would be expected to alter physical properties depending on whether they
were deposited in the normal fibrous form or as amorphous deposits. Of note
is spider drag-silk, a particularly strong silk. The gene for this has been
isolated and characterised, and the monomeric form of the protein has been
expressed in the milk of goats (Williams, 2003). The expressed protein has
been tested for fibre regeneration with the aim of producing materials with
high tensile strength (Seidel et al., 2000). The prospect of expressing the silk
gene in the wool of transgenic sheep to strengthen it was considered by the
CRC for Premium Quality Wool several years ago but it was not considered
to be sufficiently commercially viable (C. S. Bawden and G. E. Rogers,
unpublished data).
Two cell layers that play a role in the determination of the structure and
properties of the fibre are the cuticle itself and the inner root sheath of the
follicle that is involved in shaping the cuticle surface as the fibre differentiates
in the follicle. The edges where cuticle cells overlap 
(Fig. 1.6)
 favour
monodirectional fibre movement and thereby promote garment shrinkage.
No transgenic experiments have been undertaken to reduce this cuticle
behaviour but some strategies can be considered to that end, such as reducing
the edge profile to resemble the flatter profile seen in cashmere fibres. One
strategy would be to target transgenic changes to the specific proteins of the
exocuticle layer (
Fig. 1.5)
 by reducing their abundance or their level of
crosslinking, or both, or by introducing novel proteins or synthetic polypeptides
that do not crosslink. Another approach could be a disruption of the cells of
the inner root sheath cuticle cell layer that might result in a modification of
the imprinting effect of that layer on the cuticle cell edges.