WO2002029064A2

WO2002029064A2 - Method for establishing germ cell-specific gene expression

Info

Publication number: WO2002029064A2
Application number: PCT/EP2001/011354
Authority: WO
Inventors: Erez Raz
Original assignee: MERMAID PHARMACEUTICALS GmbH
Current assignee: MERMAID PHARMACEUTICALS GmbH
Priority date: 2000-10-02
Filing date: 2001-10-01
Publication date: 2002-04-11
Anticipated expiration: 2003-04-02
Also published as: AU2002221638A1; WO2002029064A3

Abstract

The present invention relates to a novel method of establishing germ cell-specific expression of nucleic acid sequences of interest in vertebrates. In particular, the invention relates to the germ cell-specific expression of marker genes, which facilitates the isolation of the germ cells and enables subsequent in vitro_ manipulations of the germ cells.

Description

Method for establishing germ cell-specific gene expression

The present invention relates to a novel method of establishing germ cell-specific expression of nucleic acid sequences of interest in vertebrates. In particular, the invention relates to the germ cell-specific expression of marker genes, which facilitates the isolation of the germ cells and enables subsequent in vitro manipulations of the germ cells.

Background of the invention

In sexually reproducing organisms primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation (reviewed in Wylie, Curr. Opin. Genet. Dev. 2000, 10:410-413). In many organisms, the primordial germ cells have to migrate from the position where they are specified towards the developing gonad where they generate gametes (Weidinger et al., Development 1999, 126(23):5295-307).

In many organisms, the identification of germ cells can only be performed in fixed, dead tissue (e.g. in chick, Tsunekawa et al. Development 2000, 127:2741-2750 and in zebrafish, Yoon et al. Development 1997, 124:3157-3165), or in organisms that permit the identification of the germ cells by generating transgenic animals carrying the GFP gene under the control of a germ cell-specific promoter (e.g. in mouse, Anderson et al. Meek Dev. 2000, 91:61-68, or trout, Yoshizaki et al. Int J Dev Biol. 2000 44:323-326.).

However, germ cells identified in fixed tissue are no longer suitable for subsequent in vitro manipulations; Furthermore, the procedures for generating transgenic animals such as DNA microinjection or mutagenesis of embryonic stem cells (ES cells) are expensive, time-consuming and not applicable for every species. Additionally, in transgenic animals the expression level of the marker gene or the number of cells expressing the marker gene would be fixed (e.g. Anderson et al. Meek Dev. 2000, 91:61-68) or not reliable (e.g. Yoshizaki et al., Int J Dev Biol. 2000 44:323-326). This represents a disadvantage in cases wherein for example the analysis of the behavior of one single cell in a group of cells is desired. Still, even if the generation of transgenic animals is possible, the early maternal expression of the transgene would generate ubiquitous expression of the marker and thus, preclude early identification of the germ cells (e.g. see Yoshizaki et al., Int J Dev Biol. 2000 44:323-326). _

The ability to identify the totipotent primordial germ cells in vivo and the subsequent isolation of the germ cells would enable in vitro manipulations of the germ cells, followed by reintroduction into the organism for purposes of transgenesis, mutagenesis and basic scientific research. However, until now the identification and isolation of primordial germ cells represents a difficult task.

It is thus an object of the present invention to facilitate the identification and isolation of primordial germ cells. This object is solved by the provision of a method for establishing germ cell-specific gene expression, preferably germ cell-specific expression of marker genes, which permits the in vivo identification of germ cells.

Description of the invention

The present invention is directed to a method for establishing specific expression of genes in primordial germ cells, wherein said method comprises the steps of (a) cloning a nucleic acid sequence of interest and a nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells into an RNA expression vector, thereby generating a fusion construct; (b) transcribing sense mRNA of the hybrid RNA of the fusion construct, and (c) introducing the mRNA of the fusion construct into an animal at an early stage of development. In a preferred embodiment, in step (a) of the method according to the invention, the nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells is further fused to a protein domain of said RNA or to a protein domain of any other RNA capable of directing specific protein degradation, subcellular localization and stability in germ cells of the resulting protein.

The fusion of protein domains which are capable of directing specific protein degradation of the fusion protein in somatic cells and localizing a viable marker (e.g. GFP) to a specific subcellular location within germ cells (in the case of this invention, perinuclear granules) enhances the specific labeling of the PGCs, if the nucleic acid sequence of interest in the fusion construct is a marker gene. Using this method, a strong contrast between somatic cells and germ cells is achieved and the label in the germ cells is characteristically granular.

The term RNA expression vector denotes a vector that includes a viral RNA polymerase such as T7, T3 and SP6.

The mRNA can be introduced into early embryos by microinjection or any other method known to the person skilled in the art.

In a preferred embodiment of the invention, the nucleic acid sequence of interest encodes a marker gene product. Particularly preferred marker gene products are selected from the group consisting of GFP (Chalfie et al, Science 1994, 263:802-805), β-lactamase (Raz et al., Dev. Biol. 1998, 203:290-294), lacZ (Lin et al., Dev. Biol. 1994, 161:77-83).

In another embodiment, the nucleic acid sequence of interest encodes a gene product of known or unknown function, including non-translated RNA molecules.

In order to prevent degradation of the mRNA of the nucleic acid of interest in the germ cells, the nucleic acid sequence of interest must be fused to a nucleic acid sequence encoding a gene or a part of a gene whose RNA is stabilized in germ cells. According to the present invention, prevention of degradation is achieved by fusing the nucleic acid sequence of interest to a nucleic acid sequence encoding a gene whose RNA is specifically stabilized in the germ cells, while being rapidly degraded in the soma.

In a preferred embodiment of the invention, the nucleic acid sequence encoding a gene whose RNA is stabilized is the vasa nucleic acid sequence or the nanos nucleic acid sequence. Nasa-like (reviewed in Raz, Genome Biol. 2000, 3:1017.1-1017.6.) and nanos- like genes (reviewed in Curr. Biol. 2000, 10:R81-3) encode proteins which are found in the germ plasm. The zebrafish vasa mRΝA can be detected initially in the cytoplasm of all cells of the embryo, with higher concentration found in four spots where the germ plasm of the zebrafish resides (Yoon et al., Development 1997, 124:3157-3165 and Knaut et al., J Cell Biol. 2000, 149:875-888). As development progresses, vasa mRΝA is specifically maintained in the region where the germ plasm resides and where the germ cells form.

In a further preferred embodiment of the invention, the nucleic acid sequence encoding a gene whose RΝA is stabilized is a vasa-analog nucleic acid sequence or a nanos-analog nucleic acid sequence.

The term "vasa- or nanos-analog" nucleic acid sequence denotes a nucleic acid sequence which retains the ability of conferring RΝA stability in germ cells. Preferably, a "vasa- or nanos-analog" nucleic acid sequence corresponds to the 3' untranslated (3'UTR) region of the vasa or the nanos nucleic acid sequence, respectively, or to a variant of the vasa or nanos nucleic acid sequence obtained by deletion, substitution, insertion or addition of nucleic acid residues.

According to one embodiment of the present invention, the gene coding for GFP was cloned into an RΝA expression vector in frame with the vasa cDΝA. The insertion of the GFP was performed into the Bglll site around nucleotide number 1178 of the vasa gene (numbering by the longer splicing form, Development 1997, 124: 3157-3165). Following linearization, mRNA synthesized from this vector was injected into 1-cell stage embryos and the fate of the RNA was tested at different stages of development using the GFP as a hybridization probe. The injected RNA was stable in the germ cells for a few days, while the RNA located in somatic tissues was rapidly degrading to undetectable level before the end of the first day of development. The fate of the RNA was determined by in situ hybridization for GFP which was gradually becoming restricted to the germ cells (see Fig. 1).

In a further preferred embodiment of the invention the nucleic acid sequence encoding a gene whose RNA is stabilized is the 3' untranslated (3' UTR) region of the vasa- or nanos nucleic acid sequence. By using deletion constructs of the vasa or the nanos encoding .nucleic acid sequence in the method according to the present invention, it was shown that more than one region of the vasa or the nanos mRNA can mediate the phenomenon of selective degradation and selective stabilization in the germ cells. One of the regions that are sufficient is the 3' untranslated region of the vasa gene, starting at nucleotide number 2222 until nucleotide 2865 (sequence numbering according to Development 1997, 124:3157-3165, GenBank Accession number AB005147). While both the vasa and the nanos 3' UTR are capable of conferring RNA stability in the germ cells, other regions of the mRNA can have similar properties as shown for the vasa gene.

The above-described method leads to specific localization of RNA in the germ cells. However, until the RNA is completely degraded in the soma, translation of the marker gene occurs and the resulting marker gene product (e.g. GFP) weakly labels somatic cells. Thus, for the purpose of specifically labeling the germ cells, the method according to the invention can be improved as follows: By fusing a protein domain capable of directing specific protein degradation in the soma as well as directing the fusion protein to a specific subcellular localization a very strong contrast between somatic and germ cells is achieved, allowing the germ cells to be identified at very early stages of development. The increased labeling specificity achieved by introducing both the RNA fusion and the protein fusion can be seen in Fig. 2. Without a fusion construct, the germ cells are not labeled with GFP. The GFP-vasa RNA fusion leads to visualization of the germ cells. An improved contrast is achieved at an early stage when a protein fusion is included in addition to the RNA fusion. Especially useful is the subcellular localization of the fusion protein which can be seen in Fig. 3.

For the purpose of the enhancement of the resolution by the protein fusion methodology, the N terminal region of vasa proved to be sufficient (bp. 1-1178 according to Development 1997, 124: 3157-3165)

These results were generalized in 2 directions. First, experiments with the zebrafish- derived construct show that labeling of the germ cells can be achieved in other organisms (e.g. Medaka fish and Xenopus). Second, another gene whose transcript is restricted to the germ plasm of zebrafish (nanos) was shown to be able to function just like the vasa 3' untranslated region in directing specific GFP expression in the germ cell of zebrafish.

The method described in this invention allows labeling of the germ cells in every injected embryo, wherein within each embryo, all germ cells are labeled. The method described herein, which enables enhanced labeling of germ cells, allows very early identification of the germi cells and can be adapted^very easily to many organisms. Compared with the available methodology of expressing GFP under the control of a germ cell-specific promoter (Yoshizaki et al., Int J Dev Biol. 2000, 44:323-326) the method described herein shows an increase in sensitivity and an improved reliability. Here, detection of the germ cells is achieved within the first 5 hours of development, while in Yoshizaki et al., the germ cells are detected at much later stages after they have finished their migration. The delayed detection time is a disadvantage, if isolation of non-differentiated early germ cells is required. While the method described herein allows consistent detection of all cells in all embryos, depending on the stage, only 30 to 70% of the transgenic fish exhibited labeling of the germ cells in the previously described method (Yoshizaki et al. Int J Dev Biol. 2000 44:323-326) . The present invention exploits the ability of the vasa mRNA or any other mRNA that exhibits similar properties for directing RNA or proteins into the germ line. The principle was proven effective using the vasa gene and the generality of the system for other germ cell specific markers was proven using the nanos gene.

The method of the present invention enables the following applications:

a. Labeling of germ cells with specific marker genes [e.g. GFP (Chalfie et al., Science 1994, 263:802-805), β-lactamase (Raz et al, Dev. Biol. 1998, 203:290-294), lacZ (Lin et al., E>ev. Biol. 1994, 161:77-83), etc.] for the purpose of folio wing the cells in living embryos, dissociating the embryo and isolating the primordial germ cells based on the expression of the marker gene (as determined e.g. by flurorescence- activated cell sorting, FACS) for downstream applications such as cell culture, RNA isolation, RNA isolation for generation of germ cell-specific cDNA libraries or hybridization probes, etc. (e.g. Long et al., Development 1997, 124:4105-4111), isolation of a cell population enriched for germ cells for establishing specific RNA or protein libraries, establishing germ cell in vitro culture.

b. Following the germ cell fate and germ cell migration in living embryos under normal conditions or in response to experimental manipulations (e.g. drug administration)

c. Expression of RNA molecules and proteins with known or unknown function (including non-translated RNA molecules) in the germ cells and assaying their effect on the cell development or behavior of the germ cells

d. Generating transgenic animals carrying the marker gene in their germ line by cloning the fusion construct downstream of a promoter that is expressed in the germ cells and subsequently, following the cells in living wild-type embryos, following the germ cells in embryos that undergo a certain manipulation, dissociating the embryo and isolating the primordial germ cells based on the expression of the marker gene (e.g. by FACS) for downstream applications (e.g. cell culture, RNA isolation etc.). While Application a.) and b.) require injection and thus, will lead to specific, but only transient expression of GFP or the RNA in the germ cells (i.e. eventually the RNA and the protein will be degraded and the progeny of the fish injected with RNA will not express the RNA in their germ cells), Application d.) is directed to the generation of stable transgenic animals.

For the purpose of generating transgenic animals, GFP or any other molecule for which expression in the germline is desired is fused to the RNA that can confer stability in the germ cells. Then, a promoter which is expressed in the germline is ligated to the fused cDNA generated in the previous step. When this construct is generated, the DNA is injected into early embryos and the progeny of these animals are screened for germline transmission of the transgene. A detailed description of cloning of a tissue-specific promoter, fusing it to a marker gene and generating transgenic animals is given in Long et al. Development 1997, 124: 4105-4111.

Application c.) is similar to Application a.) and b.), with the difference that the insert fused with the stabilized RNA possesses unknown function. A vector containing RNA sequences that can stabilize RNA in the germ cells is linearized as described above and DNA of unknown function is inserted similarly to GFP (which was described above in Application a.). As described for Application a. and b., RNA is produced, injected into an early embryo and the biological effect is monitored.

The present invention is further illustrated by the figures:

Fig 1. GFP-vasa RNA fusion was injected at 1-cell stage and the fate of the RNA was checked at different times of development by in situ hybridization using GFP as a probe. Similarly, GFP expression directed by the RNA fusion became more and more restricted to the germ cells (see middle of Fig. 2). Fig 2. RNA for GFP(top), GFP- vasa RNA fusion (middle) or GFP- vasa RNA and protein fusion (bottom) were injected at 1-cell stage and the GFP expression was followed with a fluorescent microscope. Germ cells are marked by arrowhead.

Fig 3. RNA encoding GFP -vasa RNA and protein fusion was injected into 1-cell stage embryo and the subcellular localization of the fusion protein was determined at high magnification using a fluorescent microscope.

The invention is exemplified by the following illustrative, but non-limiting example:

Example I

Germ cell-specific expression of GFP in zebrafish using the vasa coding sequence

The vasa cDNA was cloned into an RNA expression vector (pCRIItopo) and the GFP gene was cloned into the vector in frame with the vasa coding sequences. A plasmid (pCRIItopo) containing the full-length vasa cDNA {Development 1997, 124: 3157-3165, GenBank accession number AB005147) was generated by amplifying the vasa gene from zebrafish cDNA using 3' and 5' primers according to the published sequence. This plasmid was linearized using the enzyme Bglll as previously described (Sambrook et al., Molecular Cloning, 1989, CSH Press) deleting a 552bp long region flanked by two Bglll sites (between bp 1178 and bp 1730) in the coding region. The mGFP5 gene (Siemering et al. Curr. Biol. 1996, 6:1653-1663) was amplified using high-fidelity PCR (Advantage HF-PCR Kit, Clontech, according to the manufacturer's protocol) and Bglll sites were introduced upstream and downstream of the GFP by the primers used in the amplification. The insertion of the GFP was performed into the Bglll sites of the opened vector that included the vasa gene. Following linearization, mRNA synthesized from this vector (mMessage Machine Kit, Ambion, according to the manufacturer's protocol) was injected into 1-cell stage zebrafish embryos at a concentration of lOOng/microliter (as described in Westerfield, 77ze Zebrafish Book, 1993, University of Oregon Press). The fate of the RNA was tested at different stages of development using the GFP as a hybridization probe (whole mount in situ hybridization as described in Westerfield, 1993, ibid.). The injected RNA was stable in the germ cells for several days, while the RNA located in somatic tissues was rapidly degrading to undetectable level before the end of the first day of development (Fig 1). The fate of the RNA was reflected in the expression of GFP which was also gradually becoming restricted to the germ cells (Fig 2).

This invention can therefore make use of RNA molecules expressed in the germ plasm for directing RNA or proteins into the germ line. As the existence of germ plasm has also been shown in other species of commercial importance except for fish (e.g. chick, Development 2000, 127: 2741-2750.), this invention could be used in any other vertebrate species as well.

Claims

1. A method for establishing specific expression of genes in primordial germ cells, comprising the steps of

(a) cloning a nucleic acid sequence of interest and a nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells into an RNA expression vector, thereby generating a fusion construct; and

(b) transcribing sense mRNA of the hybrid RNA of the fusion construct and

(c) introducing the mRNA of the fusion construct into an animal at an early stage of development.

2. Method according to claim 1, wherein in step (a), the nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells is further fused to a protein domain of said stabilized RNA or to a protein domain of any other RNA capable of directing specific protein degradation and subcellular tocalization of the resulting protein. _ _ __

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3. Method according to claim 2, wherein the protein domain capable of directing specific protein degradation in the soma and subcellular localization and stability in germ cells is a protein domain of the zebrafish vasa RNA.

4. Method according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells is the zebrafish vasa nucleic acid sequence.

5. Method according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells is the zebrafish nanos nucleic acid sequence.

6. Method according to any one of claims 1 to 5, wherein the nucleic acid sequence encoding a gene whose RNA is stabilized in germ cells and destabilized in somatic cells is a vasa-analog or a nanos-analog nucleic acid sequence.

7. Method according to any one of claims 1 to 6, wherein the specific nucleic acid sequence encodes a marker gene product.

8. Method according to claim 7, wherein the marker gene product is selected from the group consisting of GFP, β-lactamase and lacZ.