DE102007035238A1 - Biological effect monitoring by transgenic Hydra - Google Patents

Abstract

The invention describes transgenic hydra which, in the presence of at least one stress factor in the surrounding medium, expresses at least one stress factor-inducible reporter gene, the use of this transgenic hydra as biomonitoring system and a method for detecting stress factors in samples by means of transgenic hydra. Furthermore, expression cassettes are disclosed which, after integration into suitable vectors, can be used to generate tragogenic hydra which can be used as biomonitors.

Description

The Invention relates as biomonitors, in particular for the Control of drinking water and water quality, usable transgenic Hydra, integrable in common vectors Expression cassettes for their generation and a method for Detection of pollutants by means of transgenic Hydra.

The constantly increasing amount of chemical and microbial Waste from industrial and other human activity urges the development of early warning systems based on biomonitors. To raise scientific institutions for years the demand for a biological effect monitoring, with which the effects of environmental toxins and microbial Detect germs on organisms.

Also in aquatic habitats in addition to eutrophication Environmental toxins and microbial germs are becoming increasingly common Role in the distribution and diversity of species and their survival and reproductive capacity. The mixture of environmental toxins and microbial germs in our waters is unmanageable and diverse. targeted Chemical analysis of individual substances can only ever one Gather small section of this mixture. Decisive for the survivability of the animals living there is the interaction of all existing individual substances and many other factors. So there have to be methods for detection be found that have a biological "response" to the Give mixture of environmental toxins and microbial germs to possible To recognize dangers at an early stage. Biochemistry and molecular biology Parameters are well suited for this purpose because the Time between cause (environmental poison, microbial bloom) and effect (biological effect) is short, and thus a fast and also receives sensitive answer.

Within the scope of water monitoring programs pollutants are currently measured in sediments and in water. The assessments are based on physical, chemical and / or microbiological limits. 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 referred to as "bioindicators" or "biomonitors" the presence of environmental toxins (eg, chemicals, heavy metals) and / or other stressors (eg, heat, lack of oxygen, etc.) respond, primarily by altering vital functions (eg, respiratory rate, mobility, luminescence). For example, in the DD 238 113 A1 describes the use of fish in a test channel through which the water to be tested flows. In the case of damage, the fish are driven against a grid, which is largely blocked, whereby the water level in the gutter increases. As a result, an alarm is triggered in the presence of fish damaging water constituents.

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

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

In the US 6,340,572 B1 The use of, inter alia, luminescent bacteria, fungi, fish extracts and dinoflagellates for the detection of toxic substances in a sample is used 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, such as US 4,346,070 described. In addition, the use of Hydra as biomonitor in drinking water, sewage and water monitoring z. B. from Arkhipchuk et al. Environ Pollut, 2006 Jul; 142 (2): 200-11) ("Use of Hydra for chronic toxicity assessment of water intended for human consumption"). . Pardos et al. ("Acute toxicity assessment of polish (waste) water with a microplate-based hydra attenuata assay: a comparison with the Microtox test." Sci Total Environ. 1999 Dec 15; 243-244: 141-8) and from 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) been described.

By doing U.S. Patent 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.

On a similar principle is based in the WO 99/11772 described transgenic mouse model. The transgenic mice or derived therefrom Cell cultures express a laboratory-technically detectable reporter gene (a human hormone) in the presence of toxic substances.

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) act only at relatively high concentrations of pollutants or acutely toxic substances that are 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, a 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 in the US 5,877,398 described, expressed in the presence of pollutants reporter genes, are also only bad for a continuous monitoring of drinking water and water quality or for the detection of traces of water-specific impurities, since it is not an aquatic organism, but a C. elegans non-native classical soil dwellers. 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, in terms of sensitivity, is often superior to the widely used Microtox test ( Pardos et al. "Acute toxicity assessment of Polish (waste) water with a microplate-based Hydra attenuata assay: a comparison with the Microtox test." Sci Total Environ. 1999 Dec 15; 243-244: 141-8 ). So far, however, it is not possible to separate the individual, acting on the respective biomonitor stress factors (pollutants, germs and other harmful environmental factors) against each other using either Hydra or any of the other biomonitor systems known as prior art. There is therefore a need for biomonitoring systems that are suitable for this purpose.

task The present invention is therefore a practical, sensitive and cost effective biomonitoring system for the Provide drinking water and water monitoring, the immediate information about the type of a detected stress factor gives.

The Task is accomplished by providing transgenic Hydra with the in Claim 1 listed features solved. The Subclaims give advantageous embodiments of the invention at.

Hydra is a common organism found in all aquatic habitats. The polyps live in all fresh waters and react very quickly 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 shock proteins: these responses are present in 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 specific to each different environmental signals (stress factors) such as temperature, presence of toxic Substances and microbial germs are regulated. This acts it is the Hydra genes hsp70.1 (Accession 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 changes in the water surrounding the hydrate ( Gellner et al. "Cloning and expression of an inducible hsp 70 genes in two species of hdr 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 markedly increased transcription of the hsp70.1 gene, whereby the corresponding 5'-regulatory region (promoter) of this gene is outstanding for the sensitive and specific Regulation of a reporter gene for the detection of toxic substances.

The Transcription of the hydra gene periculin, on the other hand, becomes specific by microbial water contaminants such as bacteria, fungi and Viruses or by substances characteristic of them, z. B lipopolysaccharides (LPS) from gram-negative bacteria or viral double-stranded RNA, induced. The corresponding Periculin promoter can therefore be considered very sensitive and specific Regulator for the transcription of the presence of a microbial contamination indicating reporter gene can be used.

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 NotI restriction cleavage sites and can easily be 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, SbfI, PacI, AsiSI, AscI and FseI. 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, mTFP1, 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. B. the promoter of the Hydra actin gene, as in 2 shown. 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 Sci U S A. 2006 Apr 18; 103 (16): 6208-11 ), to control the transfection efficiency and to select the successfully transfected animals. So z. B. various transgenic Hydra lines are generated, each of which is suitable for itself, on the stress-inducible expression of a suitable reporter gene and its z. B. under fluorescent light detectable gene product to indicate the presence of a particular stress factor ( 4 and 6 ). In the 1 and 2 In each case, only one stress-inducible reporter gene is shown per expression cassette. However, it is just as possible to integrate two different stress-inducible reporter genes into the expression cassette. For this purpose, reporter genes are preferably selected in this case, the gene products can be clearly distinguished from each other under fluorescent light color z. B. GFP and dsRED. The transgenic hydra produced by means of these multiply-inducible reporter gene constructs are incubated under fluorescent light when in contact with heavy metals, e.g. B. green and in the presence of bacteria z. B. glow red. This makes it possible to differentiate 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: NO 3-5.

By the use of the transgenic hydra-lines thus prepared as Biomonitoren it is possible, for example, automatic To provide systems in which the transgenic hydra is continuous be lapped by the monitored water. The corresponding hydra cultures are either permanent, preferred, however, to premature bleaching of the fluorescence avoid at regular intervals in the short term irradiated 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. about A suitable camera is the culture at each irradiation interval analyzed for the expression of the respective reporter genes. Should One of the respective reporter gene products will be detectable a corresponding analysis software generates a warning signal and / or corresponding security protocols activated (eg shutdown the water supply). Of course, the invention transgenic hydra also for the targeted study of freshwater taken water samples suitable. Here are the hydra cultures incubated with the respective water samples for a certain time and at regular intervals, as before described on the expression of the respective reporter gene products. By analogy, it is obvious, if necessary, too dissolved in water, toxicity substances to be tested with the transgenic hydra according to the invention their biological effect can be checked.

The use of transgenic hydra, which in the presence of a specific stress factor in the surrounding medium a specific reporter gene express, thus allowing the practicable, sensitive and cost-effective monitoring of the quality of drinking water and 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 in the 1 to 6 illustrated in more detail. Show it:

1 a schematic general representation of the expression vector constructs,

2 a more detailed illustration of an exemplary expression cassette,

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

4 a transgenic Hydra expressing the reporter gene GFP under regulation of the hsp70.1 promoter, in negative representation,

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

6 a transgenic hydra expressing the reporter gene dsRED under regulation of the periculin promoter.

In 1 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 an ampicillin resistance gene (Amp ^R ) as well as SP6 and T7 transcription initiation sequences as a bacterial selection marker. Via the NotI restriction cleavage site of the vector an expression cassette with two reporter genes was inserted. Furthermore, further restriction sites for the restriction endonucleases SacI, PstI (SbfI) and AscI are indicated in the diagram.

A more detailed representation of the in 1 The exemplary vector shown in FIG 2 shown. In addition to the NotI restriction sites flanking the expression cassette, further cut sites for the restriction endonucleases ApaLI, XbaI, BamHI, HindIII, PstI, SbfI, PacI, AsiSI, EcoRI, SpeI, AscI and FseI are given. Individual restriction sites used to exchange promoters and reporter genes are shown in gray. 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 constitutively expressed reporter gene (here dsRED) are also indicated. To terminate the transcription is 3 'of the respective reporter gene one of the termination region of the Hydra Ac tin gene corresponding termination sequence. 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.

In 3 An early hydra embryo is shown, 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 on the bottom right.

In 4 a transgenic hydra is shown expressing 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.

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 is common in viruses.

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 in Wittlieb et al. ("Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis." Proc Natl Acad Sci USA. 2006 Apr 18; 103 (16): 6208-11 ) described in detail. The following is a brief summary of the method:
Reporter gene constructs are used to transfect the hydra polyps. Transfection is 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 micromanipulators (Leitz, Eppendorf). Embryos become under aid of a CellTram Air pump (Eppendorf) with a micropipette. 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.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - DD 238113 A1 [0004]
• - DE 69825873 T2 [0005]
• - DE 4440320 [0006]
• - US 6340572 B1 [0007]
• US 4346070 [0008]
• US 5877398 [0009, 0011]
• WO 99/11772 [0010]

Cited non-patent literature

• Arkhipchuk et al. Environ Pollut, 2006 Jul; 142 (2): 200-11) [0008] ("Use of Hydra for chronic toxicity assessment of water intended for human consumption" Environ Pollut.
• - Pardos et al. ("Acute toxicity assessment of polish (waste) water with a microplate-based hydra attenuata assay: a comparison with the Microtox test." Sci Total Environ. 1999 Dec 15; 243-244: 141-8) [0008]
• - 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) [0008]
• - Pardos et al. "Acute toxicity assessment of Polish (waste) water with a microplate-based hydra attenuata assay: a comparison with the Microtox test." Sci Total Environ. 1999 Dec 15; 243-244: 141-8 [0011]
• - Bosch et al. "Thermotolerance and synthesis of shock proteins: these responses are present in hydra attenuate but absent in Hydra oligactis." Proc Natl Acad Sci USA, 1988 Nov; 85 (21): 7927-31 [0014]
• Gellner et al. "Cloning and expression of an inducible hsp 70 genes in two species of hdr which differ in their stress response" Eur J Biochem 1992, 210, 683-691 [0016]
• - Wittlieb et al. ("Transgenic Hydra Allow In Vivo Tracking of Individual Stem Cells During Morphogenesis." Proc Natl Acad Sci USA 2006 Apr 18; 103 (16): 6208-11 [0018]
• - Wittlieb et al. ("Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis." Proc Natl Acad Sci USA 2006 Apr 18; 103 (16): 6208-11 [0034]

Claims (19)

Transgenic Hydra, characterized in that it expresses in the surrounding medium in the presence of at least one stress factor inducible by the respective stress factor reporter gene. Transgenic hydra according to claim 1, characterized in that that the stressor is a toxic substance. Transgenic hydra according to claim 2, characterized in that that by transfecting with an expression cassette generated according to SEQ ID: NO 3 containing vector becomes. Transgenic hydra according to claim 1, characterized in that that the stress factor is microbial germs. Transgenic hydra according to claim 4, characterized in that that the microbial germs are selected from the group consisting of bacteria, fungi and viruses. Transgenic hydra according to claim 5, characterized in that that by transfecting with an expression cassette generated according to SEQ ID: NO 4 containing vector becomes. A transgenic hydra according to claims 2 and 5, characterized that these are a first when there is a first stress factor Reporter gene and in the presence of a second stress factor a second expressed from the first distinguishable reporter gene. Transgenic hydra according to claim 7, characterized in that that by transfecting with an expression cassette produced according to SEQ ID: NO 5 containing vector becomes. Use of transgenic hydra according to claim 1 to 8 as biomonitors for the detection of toxic substances and / or microbial germs. Method for detecting at least one stress factor in a sample, characterized by contacting the sample with transgenic hydra, which in the presence of at least a stress factor inducible by the respective stress factor Reporter gene express, 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 study of the thereby emitted from the reporter gene product Light. Method according to claim 10, characterized in that that the stressor is a toxic substance. Method according to claim 11, characterized in that that the transgenic hydra by transfecting with an expression cassette generated according to SEQ ID: NO 3 containing vector become. Method according to claim 10, characterized in that that the stress factor is microbial germs. Method according to claim 13, characterized in that that the microbial germs are selected from the group consisting of bacteria, fungi and viruses. Method according to claim 13, characterized in that that the transgenic hydra by transfecting with an expression cassette generated according to SEQ ID: NO 4 containing vector become. Method according to claims 11 and 14, characterized that the transgenic hydra in the presence of a first stress factor a first reporter gene and in the presence of a second stress factor express a second of the first distinguishable reporter gene. Method according to claim 16, characterized in that that the transgenic hydra by transfecting with an expression cassette produced according to SEQ ID: NO 5 containing vector become. Expression cassette characterized by a nucleotide sequence which is selected from the group consisting of the SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 nucleotide sequences. Use of an expression cassette according to claim 18 containing vector for the production of biomonitoring system usable transgenic hydra.