WO2009015623A1

WO2009015623A1 - Biological effect monitoring by means of transgenic hydra

Info

Publication number: WO2009015623A1
Application number: PCT/DE2008/001086
Authority: WO
Inventors: Thomas C. G. Bosch; Konstantin Khalturin
Original assignee: Christian-Albrechts-Universität Zu Kiel
Priority date: 2007-07-27
Filing date: 2008-06-25
Publication date: 2009-02-05
Also published as: DE102007035238A1

Abstract

The invention relates to transgenic hydra which, upon the presence of at least one stress factor in the medium surrounding it, expresses at least one reporter gene which is inducible by the stress factor; to the use of this transgenic hydra as a biomonitoring system, and to a method of detecting stress factors in samples by means of transgenic hydra. Moreover, there are disclosed expression cassettes which, after integration into suitable vectors, can be exploited for generating transgenic hydra which can be employed as biomonitors.

Description

Biological effect monitoring by transgenic Hydra

The invention relates to biomonitors, in particular for the control of drinking water and water quality, usable transgenic hydra, integrable in common vectors expression cassettes for their production and a method for the detection of pollutants by means of transgenic hydra.

The ever-increasing volume of chemical and microbial waste from industrial and other human activities urgently requires the development of biomonitor-based early-warning systems. Scientific institutions have been raising the demand for biological effect monitoring for years, with which the effects of environmental toxins and microbial germs on organisms can be demonstrated.

In aquatic habitats, in addition to eutrophication, environmental toxins and microbial germs play an increasing role in the distribution and diversity of species and their ability to survive and reproduce. The mixture of environmental toxins and microbial germs in our waters is unmanageable and diverse. Targeted chemical analysis of individual substances can only capture a small portion of this mixture. Decisive for the survivability of the animals living there is the interaction of all existing individual substances and many other factors. Thus, detection methods have to be found which give a biological "response" to the mixture of environmental toxins and microbial germs in order to detect potential hazards early.Piochemical-molecular biological parameters are well suited for this purpose because the time between cause (Environmental poison, microbial bloom) and effect (biological effect) is short, and thus one receives a fast and also sensitive answer.

Within the scope of water monitoring programs pollutants are currently measured in sediments and in water. Valuations are made on the basis of physical chemical and / or microbiological limit values. However, there are also methods that can be used to demonstrate a biological "response" to the presence of environmental toxins and pathogens in aquatic habitats, using microbial, animal or plant organisms and termed "bioindicators" or "biomimetics". which respond to the presence of environmental toxins (eg, chemicals, heavy metals) and / or other stress factors (eg, heat, lack of oxygen, etc.), essentially by altering vital functions (eg, respiratory rate, mobility, luminescence For example, the use of fish in a test channel through which the water to be tested flows is described in DD 238 113 A. In the case of damage, the fish are driven against a grid, the latter being largely blocked, thus reducing the water level This raises an alarm in the presence of fish-damaging water constituents.

DE 698 25 873 T2 describes a method for automatic biological monitoring of the water quality by evaluating the respiratory behavior and the body movement of the fish used in this case.

In DE 44 40 320, the negative chemosensory behavior of unicellular ciliates is recorded as a measure of the chemical load of environmental samples.

In US 6,340,572 Bl the use of u. a. luminescent bacteria, fungi, fish extracts and dinoflagellates used to detect toxic substances in a sample by disturbed in the presence of toxic substances, the luminescence of the test organisms and the decrease in light emission is detected against a control.

The freshwater polyp Hydra is a long established toxicological test system. So z. B. the high regenerative capabilities of this basal multicellular in the production of "artificial embryos" from cell aggregates used, which can be used in particular for the detection of teratogenic (teratogenic) toxicological effects of chemicals, as described, for example, in US 4,346,070 is also the use of Hydra as a biomonitor in drinking water, wastewater and water monitoring z Arkhipchuk et al. Environ Pollut, 2006 JuI; 142 (2): 200-11), Pardos et al. ("Acute toxicity assessment of polish (waste) water with a microplate -based hydra attenuata assay: a com- parison with the Microtox test. "See Total Environ., 1999 Dec 15; 243-244: 141-8) and by Fu et al. ("Application of the Hydra attenuata assay for identifying developmental hazards among natural waters and wastewater." Ecotoxicol Environ Saf. 1991 Dec; 22 (3): 309-19).

US Pat. No. 5,877,398 describes the use of transgenic nematodes which, in the presence of toxic substances in an environmental sample contacted with them, react with the expression of a histochemically detectable reporter gene.

The transgenic mouse model described in WO 99/11772 is based on a similar principle. In the presence of toxic substances, the transgenic mice or the cell cultures derived therefrom express a reporter gene that can be detected by laboratory tests (a human hormone).

The currently existing state of the art has a number of different disadvantages. The current limitations of the use of biomonitors arise, among other things, from a lack of knowledge from basic and applied research, the lack of pronounceability at the various levels of biological organization (cell, tissue, organism), as well as the often impractical applicability in practice. So z. B. Biomonitoring systems that are based on the analysis of respiratory rate and body movements of fish, not sensitive enough and therefore only able to detect acute toxic concentrations of pollutants. Long-term damaging effects (damage to the fetus, damage to the genome, cancer) and concentrations of pollutants which only lead to detectable symptoms after several days or weeks are not promptly recorded by these biomonitoring systems. The use in online monitoring, z. As in the production of drinking water, this is not practicable. Even the now commercially available, based on the use of luminescent organisms monitoring systems (eg Microtox assay) work only at relatively high concentrations of pollutants or acutely toxic substances, associated with a significant loss of vitality and thus luminescence loss among the test organisms. Transgenic mammalian animal models, such as the already mentioned mouse model, or primary cell cultures derived therefrom, are not suitable for continuous online monitoring of z. B. drinking water. In addition, comparatively complex laboratory analysis, possibly cell culture facilities and trained personnel are required. In addition, the production or procurement of the transgenic mice or the cultivation of the corresponding cell cultures is very cost-intensive. Transgenic Caenorhabditis elegans, which, as described in US Pat. No. 5,877,398, express reporter genes when pollutants are present, are also poorly suited for continuous monitoring of drinking water and body water quality or for detecting traces of water-specific contaminants, since it C. elegans is not an aquatic organism, but a habitat-foreign classical soil inhabitant. Continuous contacting of the test organism with the water to be monitored (advantageously, the test organism should be completely lapped by it throughout) should be impossible in the use of transgenic C. elegans, as they would drown immediately. The freshwater polyp Hydra, on the other hand, is a classic freshwater organism and is very sensitive to even the slightest traces of impurities and is therefore often superior to the widespread Microtox test in terms of sensitivity (Pardos et al., Acute toxicity assessment of polish (waste) water with a microplate-based Hydra attenuata assay: a comparison with the Microtox test "Be Total Environ., 1999 Dec 15; 243-244: 141-8), but so far it is not state of the art using either Hydra or any of the others It is therefore possible to differentiate the individual stress factors (pollutants, germs and other harmful environmental factors) acting on the respective biomonitor from one another, and there is therefore a need for biomonitoring systems which are suitable for this purpose.

The object of the present invention is therefore to provide a practicable, sensitive and cost-effective biomonitoring system for drinking water and water monitoring, which provides immediate information about the type of a detected stress factor. The object is achieved by the provision of transgenic hydra having the features listed in claim 1. The subclaims indicate advantageous embodiments of the invention.

Hydra is a common organism found in all aquatic habitats. The polyps live in all fresh waters and react very rapidly to a deterioration of the water quality (heavy metal pollution, etc.) or the water temperature with a stress response (Bosch et al., Thermotolerance and synthesis of heat shock proteins: Hydra attenuata but absent in Hydra oligactis. "Proc Natl Acad Sci USA, 1988 Nov; 85 (21): 7927-31).

The inventors have discovered genes in Hydra that are specifically regulated by different environmental signals (stress factors) such as temperature, presence of toxic substances and microbial germs. These are the Hydra gene hsp70.1 (Acces- sion No. M84019, SEQ ID NO: 1) and periculin (SEQ ID NO: 2).

hsp70.1 encodes the Hydra Heat Shock Protein (HSP) 70. This gene is very sensitive to temperature variations in the water surrounding the hydrate (Gellner et al., "Cloning and expression of an inducible hsp70 gene in two species oihydra which Differ in their stress response "Eur J Biochem 1992, 210, 683-691.) In addition, traces of toxic substances (eg, sodium azide, heavy metals) in the water lead to a significantly increased transcription of the hsp70.1 gene, resulting in the corresponding 5'-regulatory region (promoter) of this gene is outstandingly suitable for the sensitive and specific regulation of a reporter gene for the detection of toxic substances.

In contrast, the transcription of the hydra gene periculin is specifically induced by microbial water contaminants such as bacteria, fungi and viruses or by substances characteristic for them, for example lipopolysaccharides (LPS) from gram-negative bacteria or viral double-stranded RNA. The corresponding periculin promoter can therefore be used as a very sensitive and specific regulator for the transcription of a microbial contamination indicating reporter gene. The respective promoters of these genes could be identified and integrated into different DNA constructs (expression cassettes). According to the invention, these are flanked by Nelt restriction sites and can be easily integrated into a vector backbone suitable for cloning and propagation in the bacterium Escherichia coli. Each expression cassette contains unique restriction endonucleases for the restriction endonucleases ApaLI, XbaI, BamHI, Sbfl, Pacl, AsiSI, AscI and Fsel. These interfaces allow for easy replacement and combining of different promoters and reporter genes. The expression cassettes each contain at least one of the above-described stress promoters and the respective reporter gene regulated by it (eg GFP, eGFP, YFP, eYFP, CFP, dsRED, BFP, mTFPl, Emerald, Citrine, mOrange, mApple, mCherry, mGrape ). The reporter gene is followed by an expression termination sequence, preferably the hydra actin terminator. Furthermore, the expression cassette may contain another fluorescent light detectable reporter gene, which is under the control of a strong constitutive promoter. This can be z. Example, the promoter of the Hydra actin gene, as shown in Fig. 2. This additional constitutively expressible in Hydra reporter gene may optionally after microinjection of the expression vectors in early hydra embryos, as in Wittlieb et al. ("Transgenic Hydra Allow In Vivo Tracking of Individual Stem Cells During Morphogenesis." Proc Natl Acad US Pat. No. 2006 Apr 18; 103 (16): 6208-11), the control of transfection efficiency and selection of successfully transfected Thus, for example, various transgenic Hydra lines can be generated, each of which is suitable for indicating the presence of a specific stress factor via the stress-inducible expression of a suitable reporter gene and its, for example, fluorescence-detectable gene product 4 and 6), only one stress-inducible reporter gene per expression cassette is shown by way of example in each of Figures 1 and 2. However, it is just as possible to integrate two different stress-inducible reporter genes into the expression cassette In this case, reporter genes selected whose gene products under fluorescent light color can be clearly distinguished from each other kö For example, GFP and dsRED. The transgenic hydra prepared by means of these multiply-inducible reporter gene constructs are incubated under fluorescent light upon contact with heavy metals, eg. B. green and in the presence of bacteria z. B. glow red. This allows a distinction to be made between different environmental stress factors with one and the same transgenic hydra line. In multiply-inducible reporter gene constructs, it may be advantageous to dispense with the additional constitutively expressed reporter genes which serve to control the transfection success in order to avoid negative interactions with the inducible reporter genes. In this case, z. B. one of the inducible reporter genes by short-term incubation with the respective stress factor for controlling the transfection success and for the selection of the transfected animals are used. The nucleic acid sequences of the different expression cassettes according to the invention are shown under SEQ ID: N0 3-5.

By using the transgenic hydra-lines thus prepared as biomonitors, it is possible, for example, to provide automatic systems in which the transgenic hydra are continuously bathed in the water to be monitored. The corresponding hydra cultures are irradiated either permanently, but preferably in order to avoid premature bleaching of the fluorescence, at short intervals with fluorescent light of a suitable excitation wavelength. This can be z. B. with the aid of a modified fluorescence microscope or preferably a fluorescence Auflichtbinokulars done. Using a suitable camera, the culture is analyzed for the expression of the respective reporter genes at each irradiation interval. If one of the respective reporter gene products is detectable, a warning signal is generated via appropriate analysis software and / or appropriate safety protocols are activated (eg shutdown of the water supply). Of course, the transgenic hydra according to the invention are also suitable for the targeted analysis of water samples taken from fresh water. Here, the hydra cultures are incubated for a certain time with the respective water samples and checked at regular intervals, as described above, for the expression of the respective reporter gene products. By analogy with this, it is obvious that substances which may also be dissolved in water and are to be tested for toxicity can be tested for their biological effect with the transgenic hydra according to the invention.

The use of transgenic hydra, which express a particular reporter gene in the surrounding medium when a certain stress factor is present, thus allows the practical kable, sensitive and cost-effective monitoring of the quality of drinking water and bodies of water. Furthermore, the use of transgenic Hydra also allows statements on functional and structural changes at the level of cells and tissues. These include the detection of oxidative stress, pathological tissue changes, enzyme inhibition, dysfunctions of cell differentiation, genotoxic effects and much more.

The invention is illustrated in more detail in FIGS. Show it:

1 is a schematic general representation of the expression vector constructs,

FIG. 2 is a more detailed illustration of an exemplary expression cassette. FIG.

3 shows a stage of the transfection process of a Hydra embryo by means of microinjection as a negative representation,

4 shows a transgenic hydra which expresses the reporter gene GFP under regulation of the hsp70.1 promoter, in negative representation,

5 shows the induction of the hydra gene periculin by A: LPS and B: double-stranded RNA

6 shows a transgenic hydra which expresses the reporter gene dsRED under regulation of the pericinol promoter.

In Fig. 1 is a schematic general representation of the expression vector constructs is shown. The group of these expression vectors for the preparation of transgenic hydra suitable as biomonitors was designated as pHyMON by the inventors. The vector carries, as a bacterial selection marker, an ampicillin resistance gene (Amp ^R ) as well as SP6 and T7 transcription initiation sequences. Via the NotI restriction cleavage site of the vector, an expression cassette with two reporter genes was inserted. Further, in the illustration further restriction sites for the restriction endonucleases SacI, PstI (Sbfl) and AscI indicated.

A more detailed representation of the exemplary expression cassette incorporated in the vector shown in FIG. 1 is shown in FIG. In addition to the Notl restriction sites flanking the expression cassette, further cut-off cells for the restriction endonucleases ApaLI, XbaI, BamHI, HindIII, PstI, Sbfl, Päd, AsiSI, EcoRI, Spei, AscI and Fsel are indicated. Individual restriction sites used to exchange promoters and reporter genes are shown in gray. Furthermore, the position and orientation of the stress promoter and the stress-inducible reporter gene (in this case GFP) controlled by it, as well as the position and orientation of the constitutively active actin promoter and the reporter gene constitutively expressed via it (here dsRED) are indicated. To terminate the transcription, 3 'of the respective reporter gene is a termination sequence corresponding to the termination region of the hydraacetin gene. The GFP reporter gene is only expressed if a certain stress factor is present, the dsRED reporter gene product here serves to control the transfection efficiency and the selection of transgenic animals.

FIG. 3 shows an early hydra embryo which is sucked in via the fixation pipette (left) of the micromanipulator and thus fixed. The injection cannula with the vector solution can be seen at the bottom right.

FIG. 4 shows a transgenic hydra which expresses the reporter gene GFP under the regulation of the heavy metal-sensitive hsp70.1 promoter. To the medium surrounding the hydra was added 25 μmol / l cadmium chloride and the hydra incubated therein for 2 hours. The GFP expressed by the Hydra in all body cells glows bright green under fluorescent light.

FIG. 5 shows the concentration-dependent induction of the wild-type gene periculin in Hydra vulgaris by lipopolysaccharide (LPS - a characteristic cell wall constituent of Gram-negative bacteria) and after transfection with double-stranded RNA, as frequently occurs in viruses. FIG. 6 shows a transgenic hydra expressing the reporter gene dsRED under regulation of the periculin promoter. The water surrounding the hydra was added to 20 ng / ml LPS and the hydra incubated therein for 2 hours. The Hydra shines red in many places. The clonal selection has not progressed so far that the reporter gene is expressed in all body cells.

The transgenic hydra were, as described in Wittlieb et al. ("Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis.") Proc Natl Acad US Pat. No. 2006 Apr 18; 103 (16): 6208-l 1) is described in detail therefore briefly summarized:

Reporter gene constructs are used to transfect the Hyc / ra polyps. The transfection is carried out by microinjection of H. vulgaris (AEP) embryos in the 2-8 cell stage. Before the microinjection, the embryos are detached from the maternal tissue. The microinjection is performed under an inverted microscope (Zeiss Axiovert 100) and two micro-manipulators (Leitz, Eppendorf). Embryos are maintained with a micropipette using a CellTram Air pump (Eppendorf). The respective construct (0.1 μl, 0.6 μg vector / μl) is injected with a CellTram vario pump (Eppendorf). The glass needles for microinjection are made with a Vertical Pipette Puller 700 C (Kopf Instruments, Tujunga, CA). Between 20 and 30% of the embryos express the construct from day 2 onwards. After hatching, the polyps express the reporter gene as "mosaics" in small groups of cells, and clonal propagation produces polyps from such founder polyps that carry the expression construct in all cells of the body or cell line.

Claims

1. A transgenic hydra, characterized in that it has at least one

, Stress factor in the surrounding medium expressed by the respective stress factor inducible reporter gene.

2. Transgenic Hydra according to claim 1, characterized in that it is in the

Stress factor is a toxic substance.

3. Transgenic Hydra according to claim 2, characterized in that it is produced by transfection with a the expression cassette according to SEQ ID: NO 3 containing vector.

4. Transgenic Hydra according to claim 1, characterized in that it is the stress factor to microbial germs.

5. Transgenic Hydra according to claim 4, characterized in that the microbial germs are selected from the group consisting of bacteria, fungi and viruses.

6. Transgenic Hydra according to claim 5, characterized in that it is produced by transfection with a the expression cassette according to SEQ ID: NO 4 containing vector.

7. Transgenic Hydra according to claim 2 and 5, characterized in that they are in the presence of a first stress factor, a first reporter gene and in the presence of a second stress factor expresses a second from the first distinguishable reporter gene.

8. Transgenic hydra according to claim 7, characterized in that it is produced by transfection with a vector containing the expression cassette according to SEQ ID: NO 5.

9. Use of transgenic hydra according to claim 1 to 8 as biomonitors for

Detection of toxic substances and / or microbial germs.

10. A method for detecting at least one stress factor in a sample, characterized by contacting the sample with transgenic hydra, which in the

Presence of the at least one stress factor expressing a reporter gene inducible by the respective stress factor, as well as the detection of the respective reporter gene product by irradiation of the transgenic Hydra with light of a suitable excitation wavelength and the examination of the light emitted thereby by the reporter gene product.

11. The method according to claim 10, characterized in that it is the stressor is a toxic substance.

12. The method according to claim 11, characterized in that the transgenic hydra are produced by transfection with a vector containing the expression cassette according to SEQ ID: NO 3.

13. The method according to claim 10, characterized in that it is the stress factor to microbial germs.

14. The method according to claim 13, characterized in that the microbial germs are selected from the group consisting of bacteria, fungi and viruses.

15. The method according to claim 13, characterized in that the transgenic hydra are produced by transfection with a vector containing the expression cassette according to SEQ ID: NO 4.

16. The method according to claim 11 and 14, characterized in that the transgenic hydra expressing a first reporter gene in the presence of a first stress factor and in the presence of a second stress factor, a second of the first distinguishable reporter gene.

17. The method according to claim 16, characterized in that the transgenic hydra are produced by transfection with a vector containing the expression cassette according to SEQ ID: NO 5.

18. Expression cassette, characterized by a nucleotide sequence which is selected from the group consisting of the nucleotide sequences given under SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

19. Use of a vector containing an expression cassette according to claim 18 for the production of usable as biomonitoring system transgenic hydra.