Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Patent
1994-02-10
1999-10-12
Crouch, Deborah
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
800 7, 435 691, 435194, 435212, 4353201, 435325, A01K 67027, C12P 2100, C12P 2106, C12N 1500
Patent
active
059657880
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND
The present invention relates to a process for the preparation of a protein of interest in the milk of a transgenic animal. It also relates to the constructs which make it possible to obtain these animals, the animals obtained as well as the cells containing the constructs which permit the expression of a heterologous protein.
Several routes have been pursued in order to obtain proteins of biological, therapeutic or industrial interest, and which are naturally produced in small quantities or in a form difficult to purify.
It has thus been possible to produce proteins using genetic recombination techniques, by microorganisms such as bacteria or yeasts. Nevertheless, most of the proteins require, after their synthesis, a maturation stage consisting in chemical modifications of certain reacting groups, glycosylation, and the like. Prokaryotic cells do not have the adequate enzymatic content for carrying out this maturation, hence the production of inactive proteins and/or proteins with high antigenicity.
It is therefore preferable to synthesize these proteins in eukaryotic cells, which will perform the appropriate enzymatic conversions. Nevertheless, the large-scale culture of tissue cells poses a number of technical and economical difficulties.
Another approach therefore consists in causing these proteins to be produced by cells in vivo, using transgenic animals. It is desirable that the system used permits the production of proteins in large quantities and which are easily recoverable. It is therefore advantageous that the recombinant protein is produced in the mammary gland of transgenic animals, and excreted in the milk. It is indeed a biological fluid which can be easily collected, having a relatively limited complexity and a low proteolytic activity; in addition, the processes of maturation of the recombinant proteins will be probably ensured (glycosylation, phosphorylation, cleavage and the like).
SUMMARY OF THE INVENTION
Mouse or ewe mammary gland has thus been successfully made to synthesize milk proteins of another species or proteins normally absent from milk (Ref. 1 to 15).
However, the level of proteins thus produced is extremely variable. It is different from one transgenic animal to another, since it is a function of the process of integration of the transgene which is itself variable from one animal to another. The nature of the gene constructs is also essential, the elements regulating the expression of the milk protein genes being possibly many and situated at various points of the promoter region and the transcribed portion of the gene. Thus, the promoters of ovine beta-lactoglobulin, of rat WAP and of rat beta-casein are capable of causing these proteins to be synthesized in transgenic mice. The levels are, however, systematically high only in the case of beta-lactoglobulin. Likewise, the beta-lactoglobulin promoter directs the synthesis of human alpha.sub.1 -antitrypsin which reaches the value of 7 mg/ml of milk in the milk of a transgenic mouse. The alpha.sub.S1 -casein promoter permits the synthesis of human urokinase, but the promoters of the rat beta-casein and rabbit beta-casein genes used up until now are of a limited activity. The promoter of the mouse WAP gene directs the synthesis of several foreign proteins (plasminogen activator, CD4) which are secreted in the milk of transgenic mice. The quantities of proteins obtained with this promoter are however relatively small.
Furthermore, it may happen that the specificity of the promoter is modified by its association with a foreign gene. In this way Gunzburg et al. (Molecular Endocrinology 1991) obtain the secretion of growth hormone by means of a recombinant DNA under the dependence of the mouse WAP promoter, in transgenic animals; but the growth hormone is, in this case, also produced in the cerebellum, in Bergman glial cells. Such phenomena can result in toxicity and in the premature death of the animal.
The present invention is based on the demonstration of the special interest of the promoter of the rabbit WAP gen
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Devinoy Eve
Houdebine Louis-Marie
Thepot Dominique
Crouch Deborah
Institut National de la Recherche Agronomique
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