WO2002083909A2

WO2002083909A2 - Recombinant nucleotide sequence coding for a gene

Info

Publication number: WO2002083909A2
Application number: PCT/NL2002/000252
Authority: WO
Inventors: Bastiaan Jan Herman Jansen; Maurice Adrianus Monique Van Steensel
Original assignee: Multigen International B.V.
Priority date: 2001-04-17
Filing date: 2002-04-17
Publication date: 2002-10-24
Also published as: AU2002307630A1; WO2002083909A3; NL1017852C2

Abstract

The invention relates to a nucleotide sequence that codes for a gene that may be expressed at a desired moment and on a desired site in an organism into which the gene has been inserted. To this end a sequence has been integrated into an intron of the gene with the result that the gene is not expressed, or is expressed in an inactive form. The intron further comprises sequences that allow this disrupting factor to be removed, so that the gene can be expressed.

Description

Recombinant nucleotide sequence coding for a gene

The present invention relates to a recombinant nucleotide sequence coding for a gene.

The expression of a gene in a multicellular organism at a desired moment, at the desired location, is a general biotechnological problem. In order to express a gene, it is under the control of a promoter sequence, preferably under the control of an inducible promoter.

A problem frequently occurring in practice is that even when the promoter is not induced, there is uncon- trollable, poor expression (leaky expression) .

It is the object of the invention to reduce this problem and in accordance with preferred embodiments, to essentially eliminate it.

To this end the recombinant nucleotide sequence according to the invention is characterized in that it codes for a gene, which gene is under the control of a tissue- specific promoter and comprises an intron located between two exons, which intron comprises a first, second and third sequence, the first and third sequence being recognition se- quences for a recombination system that makes recombination possible, and the second sequence comprising at least one disruptive sequence selected from the group comprised of i) a transcription termination sequence for transcription, and ii) a foreign exon that optionally comprises a second dis- ruptive sequence selected from a) a transcription- termination sequence for transcription, b) a stop-codon for translation, and c) a reading frame-shifting sequence, or the complementary sequence of the recombinant nucleotide sequence. Such a construct has the advantage that should the gene be transcribed unintentionally, it will not lead to a functional ribonucleotide sequence and in particular not to a functional protein product of the gene, because of one or more of the above mentioned measures. The recombinant nu- cleotide sequence according to the invention makes it possible to permanently alter a eukaryotic organism or cell at any desired moment. The gene is under control of a tissue- specific promoter. The intron may be present in a controling sequence such as a promoter sequence of the gene, but preferably in a part of the gene read by transcription, and most preferably in the open reading frame. This renders the recombinant nucleotide sequence according to the invention particularly suitable for application in eukaryotic organ- isms such as mammals and in particular humans, various of which applications, such as medical applications, demanding an exceedingly high level of control over the (inserted) gene.

According to the invention, the recombinant nu- cleotide sequence may be an RNA or DNA sequence. The manner in which this sequence is obtained, e.g. by synthesis or genetic manipulation, is not relevant. The invention also encompasses the complementary sequence, since this can be made into a sense sequence by making use of the advantages that may be attained with the invention.

The gene may also be a prokaryotic gene not only a eukaryotic gene. In the present invention a foreign exon means an exon that is not present in the gene that codes for a functional ribonucleotide sequence of functional protein, or if the exon is found in the gene, not at that site in said gene. In accordance with the invention, the foreign exon is inserted into the gene using generally known recombination techniques. People not skilled in the art are referred to Sambrook et al. Molecular Cloning, a laboratory manual, second print, Cold Spring Harbor, New York 1989.

When reference is made in the present invention to a 'transcription-termination sequence' this obviously means a functional termination sequence on the transcribed nucleotide sequence strand of the (complementary) recombi- nant nucleotide sequence.

The recombination system is preferably a recombination system comprising at least two proteins coded by different genes, at least one of the different genes being under the control of an externally stimulatable promoter.

This deters leaky expression of the recombination system from causing excision of the intron and consequently, in the event of leaky expression of the gene or through the influence of the above-mentioned tissue-specific promoter, from expression of the gene. The (at least) two proteins may be sub units of one protein that constitutes the entire recombination system.

The recombinant nucleotide sequence preferably also comprises a second gene or complementary sequence thereof, coding for a protein of the recombination system or a subunit thereof.

In this way a single recombinant nucleotide sequence is provided, comprising the gene to be expressed un- der controlled conditions as well as the means for excising the intron. Such a recombinant nucleotide sequence is very useful as vector for genetic manipulation purposes. The recombinant nucleotide sequence preferably comprises the complete genetic code for the recombination system. If a vector comprises the recombinant nucleotide sequence according to the invention, the vector it is preferably a vector that does not integrate in the chromosome of the host. The vector preferably further possesses all the genes necessary for replication in the host cell, although these may optionally also be present on a second vector.

The first and third sequences are preferably sequences that are recognised by a recombinase.

Recombinase is very suitable as recombination system. Finally, the invention relates to a method for the genetic modification of a eukaryotic cell, characterized in that a recombinant nucleotide sequence is applied in accordance with the invention.

Therefore the second gene codes for the recombin- ase or a subunit thereof.

The invention will now be elucidated with reference to the following example. EXAMPLE Construction of a vector for the spatially- temporally controlled expression of a gene.

A possible vector according to the invention, referred to as Loxmid, is constructed from existing genetic vectors and genetic elements described in the literature. Into a vector, pUC18 or pUC19, a number of additional rare recognition sites for restriction endonucleases are introduced. These sites are important for future cloning steps. From these modified vector (s) two parent vectors are de- rived, the one named pDAD with the coding sequence for a recombinase or a sub unit thereof (for example Cre recombinase) under the control of an inducible promoter, e.g. one that is inducible with tetracycline or with a response element for vitamin A, vitamin D or another steroid hormone. The Cre gene and its inducible promoter are flanked by restriction sites that facilitate cloning in the final Loxmid. The other parent vector (named pMOM) is constructed such that it includes a gene consisting of the coding sequence for the gene of interest, comprising a native or non- native intron, under the control of a tissue-specific promoter; between two recognition sites for the recombinase that was cloned in the other vector, the intron will comprise, for example, a transcription-termination sequence for transcription, or a stop codon for translation, a reading frame-shifting nucleotide sequence, or a combination thereof. A third vector is constructed (the Loxmid, or IxCHILD) having specific cloning sites wherein, on the one hand, the coding sequence for a recombinase under control of an inducible promoter is placed, and on the other hand, the desired gene under the control of the tissue-specific promoter.

All the cloning steps are carried out using classic molecular biological techniques, supported by computer- assisted vector design, with which the expert is well ac- quainted.

The resulting vector (named Loxmid) may be used to induce gene expression in a specified tissue or cell type the moment the researcher wishes it. For example, it is possible CO U⁾ M N> h-¹ h-»

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usually associated with drug allergy. The transgenic mouse can thus serve as model for this human disorder. However, if vitamin A is applied locally, that is to say to the skin, apoptosis will only occur on the place of application. Thus the transgenic mouse is a model system for research on, for example, burn therapy. Of course, apart from Fas ligand it is also possible to use other genes in the Loxmid and the Loxmid may be adapted, depending on the model organism. For example, it may be used in basic scientific research on gene function and gene regulation in cell culture, or as remedy in gene therapy.

Claims

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recombinase or a subunit thereof.

7. A method for the genetic modification of a eukaryotic cell, characterized in that a recombinant nucleotide sequence is applied in accordance with one of the claims 1-6.