Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Patent
1994-03-07
1998-07-07
Degen, Nancy
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
4351723, 4353201, C12N 516, C12N 1506, C12N 1564
Patent
active
057767734
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to yeast artificial chromosomes and their manipulation and transfer into cells and animals, to exploit the control of gene expression, and also to the resulting cells and animals.
BACKGROUND OF THE INVENTION
The ability to transform suitable hosts with foreign DNA, and thus to express gene products not normally produced by the host, is an important goal of biotechnological research. Microorganisms can be used to produce desired proteins, while higher animals having desirable characteristics have also been produced. For example, EP-A-0264166, WO-A-8800239, WO-A-8801648, WO-A9005188 and WO-A-9211358 describe the use of lactating animals to express foreign proteins which are produced in the milk and can be isolated therefrom; this provides a very satisfactory, controlled source of pure protein.
Techniques to transfer cloned DNA into mammalian cells and transgenic animals have greatly facilitated the study of gene regulation and expression. Gene transfection experiments have also highlighted the fact that the limited size of many cloned DNA molecules prevents the efficient use of the numerous distant regulatory sequences believed to control expression.
More particularly, in order to investigate regulation of complex loci and chromosomal domains harbouring clusters of genes, it is essential to introduce very large pieces of DNA into cells and animals. The conventional approaches used in transgenic animal technology have limitations, making it difficult to introduce DNA fragments which are greater than 100 kb; see Bruggemann et al, Eur. J. Immunol. 21:1323-1326 (1991). For example, while germ line-dependent genomic imprinting may affect large areas in which a number of genes may be similarly regulated, there is no efficient method to study such "imprinted" domains. A satisfactory technique for introducing large DNA fragments would allow progress, and also facilitate the analysis of other complex loci such as the T-complex (harbouring specific deletions) for which a number of genes have been mapped which are crucial for mammalian development. In order to obtain a better understanding of the regulation of eukaryotic genes, it would be desirable to express these genes in their authentic genomic context after cloning and re-introduction into cells.
The expression of mammalian genes is controlled at various levels: the genomic context (influence of neighbouring genes), regulatory elements proximal to the exons (e.g. promoters), regulatory sequences downstream of the termination codon (polyadenylation site) and regulatory motifs further away from coding sequences (e.g. enhancers). For immunoglobulin genes, it has been shown that expression of transgenes is poor when enhancer motifs are missing, and that these can be several thousand base-pairs away (25 kb for the heavy chain 3'-enhancer) from the nearest exon.
Mouse models have been established to address the question of the immunogenicity of chimaeric, foreign and authentic antibodies used for therapeutic purposes; see Bruggemann et al, J. Exp. Med. 170:2153 (1989). It became clear that only authentic proteins escape the surveillance of the immune system.
The techniques currently used for making human antibodies involve either in vitro immunisation and immortalisation of human lymphocytes or genetic engineering. The selection of rare specific antibody-producing human lymphocytes outside the body is difficult and, once the lines are obtained, their yield and stability are poor; see Borrebaeck, Immunol. Today 9:355 (1988). Genetic engineering (also termed "humanisation" of rodent antibodies) firstly has to be done for each individual mouse or rat antibody of therapeutic use and secondly does not yield completely human antibodies; see Riechmann et al, Nature 332:323 (1988). "Humanising" existing rodent antibodies already approved for therapy is currently the most successful way to obtain less immunogenic reagents. However, it would be a considerable improvement to have a mouse strain available which makes authentic human
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Brusca John S.
Degen Nancy
The Babraham Institute
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