WO2003068971A1

WO2003068971A1 - Method of expressing an encoding sequence by xenoplus cells

Info

Publication number: WO2003068971A1
Application number: PCT/NL2003/000123
Authority: WO
Inventors: Gerardus Julianus Maria Martens
Original assignee: Multigen Holding SA
Current assignee: Multigen Holding SA
Priority date: 2002-02-18
Filing date: 2003-02-18
Publication date: 2003-08-21
Anticipated expiration: 2004-08-18
Also published as: AU2003211329A1; NL1019997C2

Abstract

The invention relates to a method of expressing a nucleotide sequence wherein a Xenopus cell is made transgenic with (i) a nucleotide construct comprising a promoter that is functionally linked to the nucleotide sequence to be expressed; or (ii) the complementary sequence of the nucleotide construct. According to the invention, the cell is made transgenic using a nucleotide construct comprising a POMC promoter, which is functionally linked to the nucleotide sequence to be expressed; or the complementary sequence of the nucleotide construct.

Description

METHOD OF EXPRESSING AN ENCODING SEQUENCE BY XENOPUS CELLS

The present invention relates to a method of expressing a nucleotide sequence wherein a cell is made transgenic with i) a nucleotide construct comprising a promoter that is functionally linked to the nucleotide sequence to be expressed; or ii) the complementary sequence of the nucleotide construct.

Such a method is known in the art. The enormous expansion of automated nucleotide sequencing has brought many genes or potential genes to light. However, the possibilities of determining the functions of such genes is lagging behind. This applies in particular to eukaryotic systems and more specifically mature or immature multicellular organisms.

It is the object of the present application to provide a method of expressing a transgene in a specific type of cell.

To this end the method according to the invention is characterised in that the cell is a Xenopus cell and is made transgenic using a nucleotide construct comprising a POMC promoter, which is functionally linked to the nucleotide se- quence to be expressed; or the complementary sequence of the nucleotide construct.

After the cells have grown into a tadpole or frog, the present invention makes it possible to bring about expression in intermediate pituitary melantrope cells. An in- teresting aspect of the invention is that the activity of these intermediate pituitary melantrope cells can be altered (increased or decreased) , without administering a non-self compound. All that is required is for the animal to be placed on a black or white surface. In contrast to induction by par- enterally administrating (i.e. injecting) an inducing compound, this can be done without any stress to the animals, especially if the animals are used to being handled. Alternatively, the surroundings of the animal can be adapted, for example using one or several electrochrome walls (including the floor) . By applying a voltage, the colour of the wall can be changed, that is to say without any physical contact with the animal. The moment in time that cell activation takes place is further very well defined. Intermediate pituitary melantrope cells can be isolated and biochemically examined using a large variety of techniques well known in the art. The present invention makes it possible to study the function of a gene in an embryonic stage (when the promoter induces expression) and in later stages of development, in which, depending on the modification of the POMC promoter (see below) , expression is made possible to a lesser or greater extent. In the present application a POMC promoter is understood to be a promoter of the prohormone "proopiomelanocor- tin" or a modified nucleotide sequence thereof being expressed in the intermediate pituitary melantrope cells only and exhibiting an activity that depends on the colour of the surface. The nucleotide sequence of this promoter is known and described in Deen, P.M.T. et al. (Mol. Biol. Evol . 9 , pp. 483-494 (1992)). In the modified POMC promoter one or more nucleotides of the POMC promoter nucleotide sequence may be deleted, replaced or inserted without loss of the typical POMC promoter activity as expressed in the tissue specificity. The modified POMC promoter according to the invention has a length of at least 100 nucleotides. The modified POMC promoter according to the invention has one or several domains exhibiting homology with the POMC promoter, which ho- mology is at least 40%, preferably at least 60%, more preferably at least 80% and most preferably at least 95%. When a construct is made of these homologous domains, it should result in a construct exhibiting substantially the same tissue specificity as the POMC promoter. The homology may be discon- tinuous, i.e. one or several non-homologous segments may be present in the POMC promoter or in the modified POMC promoter. The POMC promoter facilitates very strong expression of the nucleotide sequence. This opens the possibility of very simply, for example by deletion, limiting the POMC pro- moter activity to a level at which expression is not so strong as to inevitably disrupt the physiological processes in the cell, irrespective of which sequence is being ex- pressed.

In the present application a heterologous gene is a gene that in nature is not controlled by a POMC promoter. Usually the heterologous gene will originate from an organism other than the host cell that is being made transgenic, for example a human gene.

In the present invention the term "to bring to expression" is the subjection to transcription resulting in an RNA molecule. This may a) undergo translation resulting in a polypeptide or a functional or non-functional protein; b) hybridise with another RNA or DNA molecule, thereby disrupting the transcription or translation of the other RNA or DNA molecule; or c) form an intramolecular RNA hybrid processed by the cell and resulting in RNA interference, thereby dis- rupting the transcription or translation of the other RNA or DNA molecule.

The interested layman is asked to note that the ordinary person skilled in the art is acquainted with the fact that a cell can be made transgenic in various ways known in the art by using a nucleotide construct. The ordinary person skilled in the art is also aware of the fact that such a nucleotide construct is not necessarily a desoxynucleotide sequence, that the same may be double-stranded or single- stranded, and that in the case of a single-strand sequence, the same may be the complementary, i.e. the antisense sequence. Since the present application was written to be understood by the ordinary person skilled in the art, the interested layman may refer to standard works such as Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York, USA (1989) ) and a university at home or abroad teaching molecular biology.

According to a favourable preferred embodiment, the nucleotide construct further comprises a second promoter, which is functionally linked to an indicator gene; or comple- mentary sequence of the nucleotide construct.

In this way, without inducing the gene to be examined, it is possible to establish whether transgenesis of the cell or multicellular organism grown therefrom was success- ful. The second promoter may be any promoter with a broad tissue specificity. This could conceivably be an elongation factor (EF) l promoter for expressing the indicator gene (the marker) in all cells of an embryo, a neural tubulin pro- moter for expression of the marker in brain cells of the embryo, or a muscle promoter for expressing the marker in tail muscle cells of the embryo.

Preferably the indicator organ encodes for Green Fluorescent Protein (GFP) . In this way it is possible to establish quite simply without biochemical analysis whether transgenesis of the cell or multicellular organism grown therefrom was successful.

The POMC promoter makes it possible to specifically express the gene in the intermediate pituitary melantrope of Xenopus . Preferably a cell from the South African clawed toad frog (Xenopus laevis or Xenopus tropicalis) is used. This provides a simple model system for obtaining numerous progenies, all of which have the inserted nucleotide construct in the same site. The cell is preferably an egg cell.

This makes it possible to obtain large numbers of the above-described progenies, without having to make a non- reproductive cell omnipotent.

According to an attractive embodiment, the nucleus of a transgenic cell is removed and transferred to a cell from which the nucleus has been removed.

In this way a large number of progenies can be obtained more quickly. More in particular, the nuclei may be isolated from cells of an early transgenic embryo. These nu- clei can then be transferred to empty eggs. In this way a large number of essentially identical animals can be obtained. Since the original embryo was transgenic it is also certain that all the animals obtained will be transgenic as well . The invention also relates to a vector comprising a

POMC promoter and a nucleotide sequence that may or may not be encoding and that is under the control of the POMC promoter; or the complementary sequence of the POMC promoter and the nucleotide sequence that may or may not be encoding.

Such a vector may conveniently be used for making a cell transgenic, for example, by using the technique described by Sparrow, D.B. et al. Nucleic Acids Res. 2_8: E12 (2000) .

Finally, the invention relates to E. Coli strain DH10-B with plasmid pPOMC-GFP2, as registered with the Cen- traal Bureau voor Schimmelcultures under number CBS110047.

This strain comprises a vector affording an excel- lent starting point for the ordinary person skilled in the art for the application of the method according to the invention, wherein the expression of the nucleotide sequence is adjusted to a level that does not inevitably disrupt physiological processes in the host cell. The present invention will now be elucidated with reference to the exemplary embodiments below and the drawing in which the only figure represents a Western blot.

EXAMPLE I Materials and methods

Generation of DNA constructs

A DNA fragment of 529 base pairs containing the nucleotides -487 to +41 of the Xenopus POMC gene A promoter (wherein +1 is the transcription initiation site) was ampli- fied with the aid of PCR using the Xenopus genomic clone AXPA5 (Deen, P.M.T. et al . , Mol . Biol. Evol. 9, pp 483-494 (1992)) as a template. The primers used were pPOMC5 ' : 5 ' - ACGCGTCGACGGTACCCCGTGTAAATGTCCCTCTCC-3 ' , and pPOMC3 ' : 5 ' - TAAGAAGCTTCACTAGTCCCAAGCTGTGC-3' . This fragment was incorpo- rated in a pCSGFP2 construct (a gift from Dr. E. Amaya, Wellcome, Cambridge, Great Britain) by replacing its CMV promoter with the Xenopus POMC promoter (Sall/Hindlll fragment) obtained by PCR. The pCSGFP2 construct contains SV40 pA behind the GFP cDNA for coupling a SV40 poly-A tail to the GFP mRNA. The nucleotide sequence of the pPOMCGFP construct obtained were checked with the aid of a Big Dye Ready Reaction system (Perkin Elmer, Norwalk, CT, USA) and the pPOMC5 ' primer. With the Central Bureau for Schimmelcultures (Baarn, the Nether- lands) an E. coli strain DH10-B was registered under number CBS110047, which strain contains a plasmid of 4319 base pairs with a nucleotide construct wherein the gene coding for GFP is under the control of the POMC promoter. The gene for GFP may be replaced by cutting with the restriction enzymes BamHI and Xbal. The use of the general techniques known to the person skilled in the art such as those described by Sambrook et al. (see supra), ensures that any ordinary person skilled in the art is able to work the present invention explained in more detail below.

Preparation of unfertilised Xenopus eggs.

18 hours before the removal of eggs, mature female Xenopi (Xenopus Express, Cape Town, South Africa) were in- jected in their dorsal lymphatic cavities with 375 IU human gonadotrope hormone (hCG; Pregnyl, Organon, the Netherlands) . The eggs were deposited by the females and collected in a Petri dish. The eggs were degelled with 2% cysteine (pH 8.2) and immediately used for transgenesis .

Generation of transgenic Xenopus embryos

Sperm nuclei were purified using a Percoll gradient as described by Sparrow, D.B. et al . (supra), using digitonin for permeabilisation of sperm cells and the REMI reaction was omitted.

A Sall/Notl fragment of 1557 base pairs containing the pPOMCGFP construct and the SV40 pA signal were purified using the Qiaex II Gel Extraction Kit (Qiagen, Valencia, CA, USA) . 100 ng/2μl of this fragment were mixed with sperm nu- clei (2.5xl0⁵ in 2.5 μl "Sperm storage buffer" (Amaya E. et al. Mol. Biol. 97 pp 393-414 (1999)). Using the method described by Sparrow et al. (supra) the mixture obtained was diluted to 500 μl with "sperm dilution buffer" (Amaya E. et al., supra), and per egg ~10 nl were injected at a tempera- ture of 18 ^*C.

Normally cleaving embryos were selected at the 4- cell stage and cultured in 0.1xMMR/β% Ficoll-400 with 50 μg/ml Gentamycin at 18°C until gastrulation (stage 12) was reached. From that moment on culturing of the embryos was continued in 0. lxMMR with 50 μg/ml Gentamycin and the temperature was raised to 22°C. From stage 45 onward the tadpoles were raised in tap water at 22°C.

Selection of transgenic tadpoles

From the Nieuwkoop stage 25 onward (i.e. well before the metamorphosis to frog) , a fluorescent microscope was used to check the tadpoles for the presence of GFP fluorescence, af- ter anaesthetising living embryos with 0.25 mg/ml MS222 (3- aminobenzoic ethyl ester, Sigma) . GFP fluorescence was observed in the prosencephalon. During the further development the signal was more restricted to the forebrain. At stage 40 a clear signal was observed near the midbrain where the pi- tuitary forms. For the detection of GFP after stage 50, when the tissue surrounding the pituitary no longer allows the GFP excitation, the pituitary was isolated. In stage 56 no fluorescence was observed in any brain area but the intermediate pituitary.

EXAMPLE II

By applying the method described in Example I, transgenic frogs were generated that express a fusion protein. The fusion protein was a combination of so-called Xp24δ₂ protein and Green Fluorescent Protein (GFP) . The fusion protein was expressed in the neurointermediate lobe (NIL) . Non- transgenic (WT) and transgenic (WT) animals were placed on a white (WA) or a black (BA) surface for three weeks and then sacrificed, after which the NIL and the AL were removed. A Western blot was carried out. To this end NIL tissue material was homogenised, centrifuged and the supernatant was diluted 1:1 with SDS sample buffer (100 M Tris-HCl, 200 mM dithiothreitol, 4% SDS, 20% glycerol, and 0.2% bromophenol blue, pH=6.8), boiled for 5 minutes, and analysed with the aid of 12.5% SDS-PAGE. After electrophoresis the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) for immunodetection by means of electroblotting at 100 V for 1.5 hrs. After blocking with blocking buffer (5% w/v of low-fat milk powder in PBS, 0.5% Tween-20, pH=7.4) overnight, incubation took place with rab- bit-anti- Xp24δ₂ and Xp24δ₂-antibodies (produced in our laboratory) diluted in blocking buffer. The rabbit-antibodies were detected with horseradish peroxidase goat-anti-rabbit- labelled secondary anti-bodies according to the instructions from the manufacturer (Lumilight, Roche Diagnostics, Mannheim, Germany) and chemiluminescence was detected by means of X-ray film. For the quantification, a Biochemi system was used and the signals were analysed with Labworks 4.0 (UVP Biolmaging systems, Cambridge, UK) . Results

The fusion protein was only found in the NIL, and not in the AL (anterior lobe) , again emphasising the tissue specificity. The Western blot (Fig.) shows that in the NIL the expression of the fusion protein was 15 times higher in the black animals (placed on a black surface) than in the white animals (placed on a white surface) .

Claims

1. A method of expressing a nucleotide sequence wherein a cell is made transgenic with i) a nucleotide construct comprising a promoter that is functionally linked to the nucleotide sequence to be expressed; or ii) the comple- mentary sequence of the nucleotide construct, characterised in that the cell is a Xenopus cell and is made transgenic using a nucleotide construct comprising a POMC promoter, which is functionally linked to the nucleotide sequence to be expressed; or the complementary sequence of the nucleotide con- struct.

2. A method according to claim 1, characterised in that the nucleotide construct further comprises a second promoter, which is functionally linked to an indicator gene; or complementary sequence of the nucleotide construct.

3. A method according to claim 2, characterised in that the indicator organ encodes for Green Fluorescent Protein.

4. A method according to one of the claims 1 to 3 , characterised in that the cell is a cell from a clawed toad frog.

5. A method according to one of the preceding claim, characterised in that the cell is an egg cell .

6. A method according to one of the preceding claim, characterised in that the nucleus of a transgenic cell is re- moved and transferred to a cell from which the nucleus has been removed.

7. A vector comprising a POMC promoter and a nucleotide sequence that may or may not be encoding and that is under the control of the POMC promoter; or the complementary sequence of the POMC promoter and the nucleotide sequence that may or may not be encoding.

8. An E. Coli strain DH10-B with plasmid pPOMC-GFP2, as registered with the Centraal Bureau voor Schimmelcultures under number CBS110047.