DE10012604A1 - Transgenic animal expressing indicator gene specifically in the nervous system, useful for developing and testing therapeutic and diagnostic methods and agents - Google Patents

Abstract

Transgenic, non-human mammal (A) that contains in its somatic and germ cells at least one DNA indicator gene (I) that is expressed cell-specifically in the nervous system, is new. The gene product (II) of (I) is a marker protein for the effect of therapeutic or diagnostic agents in nervous system diseases and (I) is under control of a nervous-system specific promoter. An Independent claim is also included for a method for producing (A).

Description

The invention relates to a transgenic, non-human mammal, preferably a mouse, whose body and sex cells have an additional indicator gene contained, and the cell type-specific expressed in the nervous system. The gene became that Parasexual parent in the 1-8 cell stage of Exceed embryonic development. Areas of application of the animals according to the invention lie in the medical, pharmaceutical and biological basics and application-oriented research and diagnosis.

Transgenic animals are genetically modified animals in which at least one stranger Gene was introduced into the genome. With these animals you can Regulatory processes at the cellular level in other test systems are not Examine and influence the achieved systematic and specific way. There is also the possibility of the effects of therapeutic and diagnostic agents to test and thus the development z. B. pharmacologically active substances to test.

The production of transgenic animals is known per se. So z. B. the book by Hogan et al. Manipulating the Mouse Embryo (1994) the generation of transgenic Mice can be taken in detail. Also numerous patents for manufacturing transgenic, non-human mammals are present. In particular, US-A 4,76,866 called (crab mouse).

The object of the invention was to provide a transgenic animal which is suitable, detailed investigations of pathomechanisms to allow neurological diseases, as well as the development and testing of To enable and facilitate all kinds of pharmaceuticals. The invention is said to be new Impulses for the analysis and therapy of all kinds of neurological diseases going out.

The task is accomplished with a transgenic, non-human mammal, especially a small mammal, such as. B. a mouse or a rat, which a carries an additional so-called indicator gene. The transgenic non-human mammals are characterized in that their somatic and germ cells have at least one expressible in the nervous system DNA indicator gene carry its gene product as a detection protein for the effect of therapeutics or diagnostics for diseases of the nervous system is suitable,  the gene being under the control of a nervous system specific promoter. The indicator gene is preferably under the control of the glia-specific human GFAP (glial fibrillary acidic protein) promoters.

In a preferred embodiment, the indicator gene is a DNA sequence that encodes a fluorescent protein, in particular EGFP (enhanced green fluorescent protein) variant for the green fluorescent protein, but also DNA Sequences for blue, yellow, cyan or red fluorescent proteins (Tsien, 1998; Matz et al., 1999).

With the help of this indicator gene, the cells of the nervous system become clear marked identifiable, which plays a significant role in all injury and Play disorders of the nervous system. It is central Nervous system around the glial cell type of the astrocytes and in the peripheral nervous system around the non-myelinating Schwann cells. The involvement of astrocytes in Much has been described about diseases of the nervous system (Kettenmann and Ransom, 1995).

The indicator function of the transgene is preferred by two components achieved. On the one hand, the transgene contains a 2.2 kB fragment of the human GFAP (glial fibrillary acidic protein) promoter, a DNA sequence that expresses the expression of the downstream gene regulates (Brenner et al., 1994), of which it can be assumed that most neurological diseases lead to a lead to increased activity of this promoter (Galou et al., 1994).

On the other hand, as already mentioned, the transgene preferably contains the DNA Sequence that contains the EGFP variant of the green fluorescent protein. This Cells expressing EGFP have the advantage of being found in any conventional Fluorescence light microscope without further staining or marking techniques can be displayed.

The transgenic mammal is produced using techniques known per se, by placing the indicator gene under control of the nervous system specific promoter parasexual route in the 1-8 cell stage of embryonic development in an animal is transmitted. The wellbeing and general life expectancy of the transgenic animals are not affected by the transgenic gene products.

To date, the use of the transgenic animals according to the invention animal experiments carried out on higher animals or volunteers can be restricted. They are for new and further development therapeutic and diagnostic methods as well as the testing of pharmaceuticals  for the therapy and diagnosis of diseases of the nervous system in which Glial cells in particular astrocytes and Schwann cells as examination and Serve targets. In addition, for use for the new and Further development of therapeutic and diagnostic methods as well as testing of suitable drugs using electrophysiological methods, imaging methods for the investigation of morphology, immunohistochemical, biochemical and molecular biological methods in therapy and diagnostics of diseases of the nervous system. In particular, too living cells from the brain tissue acutely by fluorescence-activated cell sorting (FACS) can be isolated and used for testing as a high-purity cell population Find.

The fields of application of the animals according to the invention are therefore medical the pharmaceutical and biological basics and application-oriented Research and diagnosis. In particular, they are used for analysis and diagnosis of used neurological diseases. For example, a stroke, a malignant tumor or peripheral polyneuropathy.

The transgenic animals are suitable for all operations and procedures that involve the cell type-specific expression of fluorescent proteins in non-human transgenic mammals for new and further development of methods and Medicines for the diagnosis and therapy of. Nervous system disorders make useful. In particular, they are for use in electrophysiological and imaging methods suitable, the astrocyte-specific expression of fluorescent proteins like EGFP but also blue, yellow, cyan or red fluorescent proteins in non-human transgenic mammals for new and further development of methods and medicines for diagnosis and therapy of diseases of the nervous system.

The invention is then explained in more detail using examples.

Production of the transgenic animals

A 2.2 kB fragment of the human promoter of the GFAP gene (glial fibrillary acidic protein) (Brenner et al., 1994) was inserted into the multiple cloning site of the commercially available vector pEGFP (Clontech, Heidelberg, Germany) advertises. This creates a DNA construct, which the targeted expression of green fluorescent protein variant EGFP allowed in all the cells in which the GFAP promoter is active. These are astrocytes in the central nervous system (CNS) and non-myelinating Schwann cells in the peripheral nervous system (PNS). Transgenic mice were linearized by pronuclear microinjection DNA fragment from the GFAP promoter, the coding sequence of the EGFP and a polyadenylation site in fertilized oocytes from FVB / N-  Tribe generated. The transgenic animal was genotyped by Polymerase chain reaction with oligonucleotide primers from the EGFP sequence.

Characterization of the transgenic animals

Transgenic animals of mouse line 732 have been detailed in terms of their transgenic Expression characterized. For this purpose, morphological and immunohistochemical as well as electrophysiological examinations.

Astrocytes in all brain regions, including Bergmann 's glial cells Cerebellum and the Müller cells of the retina were characterized by high EGFP Expression in the cytoplasm. EGFP expression was possible in both conventional fluorescence microscope at all magnifications as well as with a confocal laser scanning microscope can be observed. In the PNS, myelinating Schwann cells based on their EGFP-mediated fluorescence and their characteristic morphology can be recognized. An immunohistochemical Analysis with antibodies directed against a neuronal protein (NeuN) and against Oligodendrocytes (myelin-associated glycoprotein) showed that the green fluorescent cells are not neurons or oligodendrocytes. A positive one Immunostaining when using antibodies against GFAP identified the green fluorescent cells clearly as astrocytes. Patch clamp investigations of the green fluorescent cells in acute tissue sections of different brain regions transgenic animals showed clear ion channel expression patterns of astrocytes, which were indistinguishable from wild-type astrocytes. Under the use of Calcium chelating fluorescent dyes such as Calcium Orange have managed to transmitter induced changes in cytoplasmic calcium concentration in EGFP-expressing astrocytes of transgenic mice and visible to do. The green fluorescence of the astrocytes could be exploited to be in situ isolate differentiated cells using fluorescence-activated cell sorting (FACS). For this purpose, single cell suspensions from mechanical and enzymatic crushed brain tissue of transgenic mice. To FACS isolation, RT-PCR analyzes could be performed.

In transgenic mice there was an increased expression of the green fluorescent Protein observed after a stab injury in the cortex. This experiment shows that the EGFP- observable and measurable via the fluorescence intensity Expression is a direct parameter of cell activation. In these transgenic animals it becomes possible for the first time via the fluorescence intensity and its change in entire nervous system, in a selected brain region or on the plane single cells directly activate cells in living tissue without prior Staining techniques solely by observation with a fluorescence microscope or other fluorescence intensity measuring devices in real time and to measure up. This direct observation also allows for living tissue standardized and quick search and testing of new substances that are based on diverse reaction mechanisms in the nervous system can influence. As  Measurement parameters are the changed EGFP expression, calcium concentration and Changes in membrane conductivity but also changes in gene activity (RT-PCR) Available. Numerous models of damage are ideal for the mouse as an ideal laboratory animal of the nervous system. These animal experiments can be found here described transgenic animals are carried out and analyzed. In Hereditary diseases and their pathomechanisms can be examined to an increasing extent become. By crossing the indicator gene in mouse models more human Hereditary diseases can involve astrocytes beyond the ones described above Expression of a fluorescent protein can be analyzed.

literature

Brenner, M., Kisseberth, WC, Su, Y., Besnard, F., and Messing, A. (1994). GFAP promoter directs astrocyte-specific expression in transgenic mice. J. Neurosci. 14, 103-1037,
Galou, M., Pournin, S., Ensergueix, D., Ridet, JL, Tchelingerian, J., Lossouarn, L., Privat, A., Babinet, C., and Dupouey, P. (1994). Normal and pathological expression of GFAP promoter elements in transgenic mice. Glia 12, 281-293.
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo: A Laboratory Manual. Spring Harbor Laboratory; ISBN: 0879693843.
Kettenmann, H. and Ransom, BR (1995). Neuroglia., H. Kettenmann and BR Ransom, eds. (New York: Oxford University Press).
Matz, MV, Fradkov, AF, Labas, YA, Avitsky, AP, Zaraisky, AG, Markelov, ML and Lukyanov, SA (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969-73.
Tsien, RY (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509-44.

Legend to the illustrations

Fig. 1: Plasmid map of the DNA vector used to produce the transgenic animal.

Fig. 2: Nucleic acid sequence of the linearized DNA fragment used for the microinjection. The promoter region is highlighted in gray, the EGFP gene is framed.

Fig. 3: Wild-type GFAP and GFAP-promoter-controlled EGFP expression are colocalized in astroglial cells from different brain regions. Brain sections from the cerebellum (A, B) and cortex (C, D). In A and C the green fluorescence of the EGFP is shown in the cells, in B and D an immunohistochemical staining against GFAP.

Fig. 4: EGFP-expressing astrocytes can be easily recognized in acute isolated living brain sections and characterized electrophysiologically. An astroglial cell was selected in A and characterized by patch clamp analysis (inset in B). During the electrophysiological measurement, this cell was filled with the red fluorescent dye Texas Red via the patch pipette. It can be shown directly which cell from A was examined. C, D and E show two more sample cells. The current profile in D is characteristic for a mature glial cell, that in E for a young one.

Fig. 5: Brain sections with green fluorescent astrocytes (A) can be loaded with the calcium-chelating fluorescent dye Calcium Orange-Acetoxymethylester (B). Then transmitter-induced changes in calcium concentration (C and D) can be measured. In this example, norepinephrine was used.

Fig. 6: In GFAP / EGFP transgenic mice, EGFP is also expressed in non-myelinating Schwann cells. Cryosections of a sciatic nerve A and B in longitudinal section, C and D in cross section. The green fluorescent non-myelinating Schwann cells are shown in A and C. In B and D phase contrast images of the tissue sections.

Claims (9)

1. Transgenic, non-human mammal, characterized in that its somatic and germ cells carry at least one cell type-specific expressible DNA indicator gene, the gene product of which is suitable as a detection protein for the action of therapeutics or diagnostics in diseases of the nervous system, the gene under is under the control of a nervous system-specific promoter. 2. Transgenic mammal according to claim 1, characterized in that the Indicator gene under the control of the glia-specific human GFAP promoter stands. 3. Transgenic mammal according to one of claims 1 or 2, characterized characterized in that the indicator gene is a DNA sequence that a encodes fluorescent protein. 4. Transgenic mammal according to claim 3, characterized in that the DNA Sequence encodes the EGFP variant for the green fluorescent protein. 5. Transgenic mammal according to one of claims 1 to 4, characterized in that it is a mouse or coffee. 6. A method for producing a transgenic mammal according to one of the Claims 1 to 5, characterized in that the indicator gene is under control of the nervous system-specific promoter by parasexual route at the latest in Eight cell stage of embryonic development is introduced into the animal or into his ancestors were brought in. 7. Use of a transgenic mammal according to any one of claims 1-5 for the New and further development of therapeutic and diagnostic methods as well for testing drugs for the therapy and diagnosis of diseases of the nervous system, in which glial cells in particular astrocytes and Schwann- Serve cells as objects of investigation and target. 8. Use of a transgenic mammal according to any one of claims 1-5 for the New and further development of therapeutic and diagnostic methods as well for testing suitable drugs in which electrophysiological Methods, imaging methods for the investigation of morphology, immunohistochemical, biochemical and molecular biological methods at  Therapy and diagnostics of diseases of the nervous system used become. 9. Use of a transgenic mammal according to any one of claims 1-5 for the New and further development of therapeutic and diagnostic methods as well for testing suitable drugs by using highly purified cells a specific cell population from the brain tissue acutely by fluorescence activated cell sorting (FACS) can be isolated.