The targeting of a recombinant protein to the milk of a transgenic animal (Figure 1) is achieved by first generating an expression
vector containing the gene encoding the protein of interest fused to milk-specific regulatory elements. This transgene is
then introduced in the germline of the chosen production species. Pronuclear microinjection of one-cell embryos (Figure 1)
or, alternatively, transfection into a primary cell population suitable for somatic cell nuclear transfer (Figure 2) have
both been used to generate transgenic founders.
Figure 2. Schematic representation of the somatic cell nuclear nuclear transfer process employed for the production of transgenic
animals used for the production of recombinant proteins.
Following germline integration, mammary gland-specific transgenes are predictably inherited by the offspring of the founder
animal. The expression level of the protein(s) of interest is variable. Concentrations surpassing 1 g/L are attained routinely
and levels of up to 20 g/L have also been achieved. Expression levels are dependent on the mammary-specific regulatory sequences
employed, the gene expressed, and the integration site of the transgene. Milk can easily be obtained using established large-scale
technologies of the dairy industry, and is an excellent starting material from which recombinant therapeutic proteins can
be purified. The choice of the production species is largely driven by the expected quantity of the therapeutic protein needed.
There is usually a trade-off between milk yield and time to natural lactation. Another consideration may be a species-specific
ability to perform specialized post-translational modifications more efficiently.
Figure 3. Timeline associated with the creation of a herd of transgenic goats producing recombinant proteins in their milk.
SCREENING THE MAMMALS
Transgenic mice have mainly been used for the testing of expression constructs prior to or concomitant with the generation
of larger founder transgenic animals. This model allows the relatively inexpensive and rapid evaluation and optimization of
transgene constructs and has proven crucial to the development of milk expression technology. The model allows the definition
of regulatory sequences that efficiently target expression of heterologous genes to the mammary gland.7 Obviously, the very limited milk yield from transgenic mice restricts expression of recombinant proteins to small amounts.
But this can be sufficient to obtain meaningful data on the protein of interest. As an example, it was possible to purify
enough Malaria antigen MSP142 from transgenic mouse milk to test for immune protection in a primate model.8
The generation of transgenic rabbits by pronuclear microinjection is straightforward and inexpensive. Relative to ruminants,
rabbits have a short gestation interval that allows up to eight lactations per year. However, only 1.5 L of milk can be obtained
per lactation, and this limits the value of this expression system to products with a commercial scale in the low-kilogram
range;9–12 Labor-intensive milking and high husbandry costs could become prohibitive for larger quantities of purified proteins.
Recombinant protein production in the milk of transgenic sows has been reported for human Protein C,13 factor VIII,14 and factor IX.15 Lactating sows can yield a surprising amount of milk (100–200 L) and it has been reported that the porcine mammary gland
cells can carry out the complex post-translational modifications (γ-carboxylation, proteolytic processing) on factor IX and
Protein C at rates higher than those encountered with mammalian cell and transgenic mouse milk systems.16