Transgenic animal bioreactors |
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Authors: | Houdebine Louis Marie |
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Institution: | (1) Unite de Biologie du Développement et Biotechnologie, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France |
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Abstract: | The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize
proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk,
egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant
proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in
the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although
the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected
somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried
out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is
clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from
the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in
poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC
vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene
expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and
introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved,
carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular
machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes
involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically
in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the
recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves.
This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from
milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule
such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification
of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous
proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins
are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic
animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on
one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may
reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used
as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate
some mammary infectious diseases.
This revised version was published online in August 2006 with corrections to the Cover Date. |
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Keywords: | Recombinant proteins transgenic animals milk |
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