Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Patent
1996-01-25
2000-11-21
Degen, Nancy
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
435463, 435352, C12N 1585
Patent
active
061501697
DESCRIPTION:
BRIEF SUMMARY
This application claims priority from International application PCT/GB94/00849, filed Apr. 21, 1994.
This invention relates to DNA constructs for inserting heterologous gene sequences into a host genome so as to obtain expression of the heterologous gene, to methods of inserting heterologous gene sequences into a host genome and to organisms carrying modified host genomes.
In one particular aspect this invention relates to constructs for inserting a heterologous gene into an endogenous gene in a host genome so that the heterologous gene is expressed in place of or in addition to the endogenous gene. In a second particular aspect this invention relates to methods for functionally integrating a heterologous gene sequence (transgene) into a specified gene of a host genome so as intimately to couple transgene expression with the endogenous transcriptional and post-transcriptional regulatory elements, to constructs for use in said methods, and to genetically modified cells and transgenic animals generated with such constructs and their descendants.
Genetic engineering involves the fusion of different gene sequences. In many cases this is performed with the intention of expressing a heterologous gene sequence in a fashion which is identical to or in part reflects the expression pattern of another gene. To achieve the desired expression level, distribution and/or timing or the sequence being expressed, regulatory sequences of the gene being copied are fused with the sequences of the gene which is to be expressed to generate an expression construct. However, in many applications involving higher eukaryotic cells, such as the selection of particular stem cells or the production of heterologous proteins from transgenic animals, it is extremely difficult to generate an expression construct whose pattern and level of expression adequately mimics those of the gene being copied.
It is known to introduce heterologous genes into mammalian cells including stem cells, transgenic animals and in vitro maintained cell lines. However, despite specific design, existing expression constructs, when integrated into the host genome, rarely provide the desired level and distribution (both spatial and temporal) of gene expression. Expression constructs are known that attempt to mimic the expression profile of an endogenous gene by incorporating known regulatory elements of the endogenous gene. However, success with these constructs is low partly because functional detail of the endogenous gene structure including the location and identity of such elements and the contribution each component makes in regulating gene expression, for the most part, remains unknown. Other problems are associated with randomly integrating expression constructs including positional effects of the site of integration and random mutation of endogenous gene expression.
Furthermore, to position and define regulatory elements in endogenous genes, often at some distance from the transcribed region of the gene, often demands much painstaking work. The distal positioning of these elements is also often important to their function and may be difficult to reproduce in transgenic expression constructs.
Further still, having identified and engineered the endogenous regulatory elements into heterologous gene expression constructs, there is little assurance that any particular transgenic expression construct will function correctly once introduced at random into the genome.
Early attempts to produce heterologous proteins in transgenic animals principally focused on the use of transgene constructs comprising promoter regions derived from one gene fused to cDNA coding sequences from another gene. For the most part the fusion constructs function poorly, if at all, and the level of expression obtained is far lower than that of the endogenous gene.
This is in contrast with intact genes, such as the ovine whey protein betalactoglobulin (BLG). High-level expression of the encoded protein is obtained in transgenic mice harbouring a full-length BLG gene complete with all introns
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Lathe Richard Frank
Mountford Peter Scott
Smith Austin Gerard
Degen Nancy
The University of Edinburgh
Yucel Irem
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