DE19829660C1 - Transgenic non-human mammal useful for testing compounds for anticancer activity - Google Patents

Abstract

A non-human mammal, comprises a gene, that codes for a SCUZ protein, that is altered so that it is switched off, or can be switched off in a time- or tissue-specific manner. Independent claims are also included for the following: (1) a cell, from a non-human mammal as above; (2) a method to prepare a non-human mammal as above; and (3) DNA comprising a 9312 bp sequence, fully defined in the specification, or a sequence, that isn't cDNA, that differs by one or more bases and which can hybridize with the DNA.

Description

The present invention relates to a non-human mammal in which a is changed for a gene coding for a protein, the protein being an SRCR, contains a CUB and a ZP domain. The invention further relates to a ver drive to manufacture such a mammal and its use for Testing of substances influencing the protein.

The term "SRCR" domain means "scavenger receptor cysteine rich" Domain. Such a domain comprises about 110 amino acids and is found in many proteins that are involved in elementary processes of the cell, e.g. B. Cell Difference tion, cell-cell contact or immune response, or as receptors act. Furthermore, the terms "CUB" domain and ZP "domain each have a defined protein range for protein / protein interactions important is.

DE 197 30 997 C1 is a human protein known that contains an SRCR, a CUB and a ZP domain. His gene will labeled with the DMBT1 gene and mapped onto chromosome 10q25.3-q26.1. It is a tumor suppressor gene found in brain tumors, e.g. B. at 10% of the medullobla stome and 25% of glioblastomas, as well as homozygous in breast tumors (13%) Has deletions. Furthermore, the carcinogenesis of lung carcinoma noms, from melanoma, pancreas, endometrial, esophagus, colon and Gastric carcinomas as well as others have been linked since at around 40-50 % of all tumors have chromosome 10q losses. Furthermore are Genes from the mouse (CRP-ductin gene) and the rat (Ebnerin gene) are known to each encode a protein that has an SRCR, a CUB and a ZP domain contains. Proteins containing such domains are shown below SCUZ proteins called.

However, the exact function or mode of action of SCUZ proteins is not yet known.

The present invention is therefore based on the object of providing a means with which this can be investigated. A means should also be available with which substances can be tested that have an influence have on SCUZ proteins.

According to the invention, this is the subject of the claims reached.

It has been recognized that the function or mode of action of an SCUZ protein can be investigated by means of a non-human mammal in which a gene which codes for an SCUZ protein is changed in such a way that it is either switched off or time or can be switched off in a tissue-specific manner. Such a mammal was generated by homologous recombination using a vector construct comprising the promoter and exon 1 of the CRP-ductin gene flanked by IoxP sequences (see FIG. 2).

According to the invention, the knowledge is used, a non- human mammal in which a gene for an SCUZ Protein encodes, is changed so that it is switched off or time or can be switched off in a tissue-specific manner.

The term "non-human mammal" includes any mammal in which a gene can be modified that codes for an SCUZ protein. Examples of such Mammals are mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, Pig, dog and cat, with mouse being preferred.

The term "an SCUZ protein" includes any protein that contains at least one Contains SRCR, a CUB and a ZP domain. The protein can be or modified form, e.g. B. as a fusion protein or as a fragment, the modified form also comprising a modified amino acid sequence. Furthermore, the protein can be homologous to the non-human mammal or be heterologous. In the latter case, it may be convenient if that goes with that  Mammal homologous protein is turned off. It can also affect the mammal heterologous protein from another mammal or human or a other organism, e.g. B. the fruit fly. Preferably that is the above protein is a human, e.g. B. that encoded by the DMBT1 gene Protein, a mouse, e.g. B. the protein encoded by the CRP-ductin gene, or a Rat protein, e.g. B. the protein encoded by the Ebnerin gene.

The expression "modified gene" indicates that a gene that is responsible for a SCUZ protein encoded, is changed so that it is switched off or time- or tissue-specific can be switched off. The former can e.g. B. thereby be achieved that the gene has an insertion or deletion, whereby the Gene no longer encoded for the functional protein. As an insertion z. Legs Selection cassette, such as neoTK, are available. Furthermore, z. B. an exon or part of it, like the promoter and exon 1 of the CRP-ductin gene, are missing. Furthermore, a time or tissue-specific switching off of the gene z. B. be achieved in that it is under the control of an inducible or tissue specific promoter. The gene can also have sequences which can be recognized by a recombinase, whereby after their activation a or multiple parts of the gene are excised, resulting in a deletion leads. Such sequences are e.g. B. IoxP sequences by a recombinase Cre can be recognized. The activation of the recombinase can be done by different Measures take place. For example, the recombinase gene can be found under the Control of an inducible or tissue-specific promoter and already exist in the mammal in which the above gene is switched off shall be. The recombinase gene can also be the mammal or its Descendants are fed, this z. B. by conventional transfection procedure or by crossing with another mammal that is the recom contains binase gene, can be done.

In a preferred embodiment, a non-human mammal according to the invention has the allele shown in FIG. 3A. A non-human mammal which has the allele indicated in FIG. 4C, 1st or 4C, 2nd is particularly preferred. It is very particularly preferred if the non-human suckling animal is homozygous for the above allele.

Another object of the present invention are cells that from the above non-human mammal. These cells can be in any form, e.g. B. in a primary or long-term culture.

A non-human mammal according to the invention can be generated by conventional methods. A procedure is favorable which comprises the following steps:

• a) Transfection of embryonic stem cells from a non-human Mammal with a vector construct that recombines with the DNA allows the embryonic stem cells, the recombination leads to a modification of a gene which codes for an SCUZ protein, and the modification is such that the gene is derived from a recombinase has recognizable sequences,
• b) isolation of cells transfected in (a) and treatment of these with a Recombinase and introduction of the cells into female animals of a non- human mammal,
• c) Analysis of the offspring obtained in (b) for a gene which is suitable for a SCUZ Protein encodes, the gene being modified such that it is switched off is or can be switched off in a time-specific or tissue-specific manner, and possibly
• d) treatment of the offspring analyzed in (c) with recombinase.

Regarding the terms "non-human mammal", "an SCUZ protein" and "modified gene" is referred to the above statements. Furthermore, the person skilled in the art knows conditions and materials in order to carry out steps (a) - (d)  perform.

Furthermore, the term "embryonic stem cells" refers to any embryo The stem cells of a non-human mammal that are changing gene that encodes an SCUZ protein. Preferably, the embryonic stem cells from the mouse, especially cells E14 / 1. Em bryonic stem cells that contain a modified gene that is responsible for a SCUZ Protein encodes, the modification being such that the gene from a Re combinease recognizable sequences are also the subject of present invention.

The term "vector construct" includes any vector construct that is represented by Recombination with the DNA of embryonic stem cells for a change of a gene coding for an SCUZ protein, the change is such that the gene is switched off or time or tissue-specific can be turned off. For example, the vector construct can be a DNA having an exon or part thereof of the above gene men with a selection marker, these elements of sequences flanked, which are recognized by a recombinase. Preferably includes the vector represents the promoter and exon 1 of the CRP-ductin gene together with the neoTK selection cassette, these elements flanked by IoxP sequences are. Such a vector was used at the DSMZ (German collection by Mikroor ganisms and cell cultures) as plasmid CRP-ductin-KOK-creIoxP under DSM 12192 on May 27, 1998.

Another object of the present invention is a nucleic acid, in particular DNA, which comprises the promoter and exon 1 of the CRP-ductin gene. Preferably, the DNA comprises the sequence of FIG. 1 or a sequence different therefrom by one or more base pairs, the latter sequence hybridizing with the sequence of FIG. 1 and not being a cDNA. The DNA of FIG. 2, ie the insert DNA of the plasmid CRP-ductin-KOK-creIoxP, is particularly preferred.

The term "hybridization" indicates hybridization under conventional ones Conditions, especially at about 20 ° C below the melting point of the Hybridization sample used DNA.

A nucleic acid according to the invention, in particular DNA, can be used as such or in the form of a vector, in particular expression vector. Vectors are known to the person skilled in the art. A nucleic acid according to the invention, ins special DNA containing vector is therefore also the subject of the present the invention.

The present invention provides a non-human mammal is in which a gene is changed, which codes for an SCUZ protein, the Change is such that the gene is switched off or time or can be switched off in a tissue-specific manner. With such a mammal or Cells from this can affect the function or mode of action of an SCUZ protein be searched. It is also possible to determine the relationship between a switched off or modified SCUZ protein and the most advanced Investigate diseases, especially carcinomas. Such karzino me are z. B. brain tumors, breast tumors, lung carcinomas, melanomas, pan kreas, endometrial, esophageal, colon and gastric carcinomas. Furthermore is it is possible to test substances with which the protein or its function or mode of action can be influenced. This also includes testing Substances with which the function or mode of action of the protein at least can be partially replaced. The present invention thus also provides the Basis to develop active substances with which the above diseases, can be met. It also provides genomic sequences of the CRP ductin gene, which contributes to the further understanding of genes that are responsible for a Encode SCUZ protein. These sequences also give the opportunity to add funds develop with which the detection of such genes enables or in the Ex genes can be interfered with. Furthermore, the present delivers Invention non-human mammals, e.g. B. sheep, cows, etc., in which large Amounts of SCUZ proteins, especially those made by humans  can be.

Brief description of the drawings.

Fig. 1 shows a DNA of the invention comprising the promoter and exon 1 of the CRP-ductin gene.

Fig. 2 shows a DNA of the invention which is present as an insert in the plasmid CRP ductin COC creIoxP. The DNA comes from the DNA of FIG. 1, ie it comprises the entire sequence (bp1-9312) of the DNA of FIG. 1. In this fragment, starting from the base pair 4114, there is an IoxP sequence and an additional HindIII and Spell Interface comprehensive insert of about 74 bp inserted. Furthermore, the DNA from base pair 5336 has a sequence insert of approximately 3.0 kb, which comprises a neoTK selection cassette with flanking IoxP sequences.

Fig. 3a-c shows the homologues recombination event between the fiction, modern DNA of Fig. 2 and the CRP gene or the ductin is obtained from genotype.

Fig. 4a-c shows homologous recombination (1) and (2) after the occurrence of the genotype of Fig. 3 a Cre recombinase treatmen has been subjected to development.

The invention is illustrated by the example.

Example 1: Generation of a non-human suckling device according to the invention animals

A mouse is generated in which the CRP-ductin gene is changed. For this purpose, a vector is used which contains the DNA from FIG. 1. This fragment has exons 1 and 2 of the CRP-ductin gene and its promoter. Furthermore, the fragment is modified in that from base pair 4114 it has an IoxP sequence and an additional HindIII and Spel cleavage site of approximately 74 bp. Furthermore, the fragment from base pair 5336 has a sequence insert of approximately 3.0 kb, which comprises a neoTK selection cassette with flanking IoxP sequences (cf. FIG. 2).

This vector is introduced into embryonic mouse stem cells E14 / 1 by electroporation. This wears a brown fur. Stably transfected cells are obtained by selection with 350 µg / ml G418. Genomic DNA is isolated from these cells, digested with HindIII, electrophoretically separated and blotted. For the hybridization, a labeled DNA fragment, probe A, from FIG. 3 is used, which is flanking the 5 ′ end of the region that is suitable for homologous recombination on the left-hand side. E14 / 1 cells are identified which are heterozygous, ie which contain a wild-type and a recombined allele (cf. FIGS. 3 and 4). These cells are transiently transfected with 5-30 µg of a Cre expression plasmid, e.g. B. pMC-Cre, before 1 µM Gancyclovir is added. This is used to select cells that have lost the neoTk selection cassette. Recombination event 1 or 2 is present in these cells (cf. FIG. 4). The former has a deletion of the neoTk selection cassette and is referred to as a "floxed" allele. In hybridization with probe B, this shows an additional 2.1 kb spel band in addition to the approximately 15 kb wild-type spel band. Recombination event 2 has a deletion of exon I and the neoTK selection cassette and is referred to as a "zero" mutant. In hybridization with probe B, this shows a 4 kb wild-type BglII band and an additional 2.9 kb BglIII band. Alternatively, a 15 kb wild-type spel band and an additional 13.5 kb spel band are also shown (see FIG. 4).

The cells obtained are injected into C57BL / 6 mouse blastocysts. This wears a black fur. The blastocysts (12-15) turn into a false swan The mouse, B6D2-F1, is transferred and chimeric, black-brown mice become  receive. Chimeric females are crossed with C57B1 / 6 males, thereby the preservation of offspring is ensured. Those with brown fur will be analyzed by Southern blotting and / or PCR, thereby identifying mice that are heterozygous for the "floxed" allele or the "zero" mutant. By crossing heterozygous mice, homozygous mice are obtained Those that are homozygous for the "floxed" allele become a cross with mice that have a Cre recombinase gene under the control of a tissue-specific promoter included. Mice are obtained which are homo zygot for the "floxed" allele and heterozygous for the Cre recombinase gene are.

It turns out that a non-human mammal according to the invention in which a gene that codes for an SCUZ protein can be switched off is or can be switched off in a time-specific or tissue-specific manner.

Claims (21)

1. Non-human mammal that has a gene that is responsible for an SCUZ protein encoded, is changed such that it is switched off (a) or time or can be switched off in a tissue-specific manner (b). 2. The non-human mammal of claim 1, wherein the gene is a CRP- is the ductin gene. 3. The non-human mammal of claim 1, wherein the gene is a DMBT1 gene. 4. The non-human mammal of claim 1, wherein the gene is a level rin gene is. 5. The non-human mammal of claim 1 or 2, wherein the CRP- ductin gene in (a) has a deletion that contains the promoter and exon 1 includes. 6. The non-human mammal of claim 1 or 2, wherein the CRP- ductin gene in (b) comprises IoxP sequences flanking the promo tor and exon 1 are present. 7. A non-human mammal according to claim 1, 2 or 5, wherein the mammal has the allele of Fig. 4C, 2.. 8. A non-human mammal according to claim 1, 2 or 6, wherein the mammal has the allele of Fig. 4C, 1.. 9. Cells obtained from the non-human mammal according to one of the  Claims 1-8. 10. A method of providing a non-human mammal according to claim 1 comprising the steps of:

• a) Transfection of embryonic stem cells of a non-human mammal with a vector which enables recombination with the DNA of the embryonic stem cells, the recombination resulting in a modification of a gene which codes for an SCUZ protein and the modification in such a way is that the gene comprises sequences recognizable by a recombinase,
• b) isolation of cells transfected in (a) and treatment of these with a recombinase and introduction of the cells into female animals of a non-human mammal,
• c) Analysis of the progeny obtained in (b) for a gene which codes for a SCUZ protein, the gene being modified such that it is switched off or can be switched off in a time-specific or tissue-specific manner, and if appropriate.
• d) treatment of the offspring analyzed in (c) with a recombinase.

11. The method of claim 10, wherein the gene is a CRP-ductin gene. 12. The method of claim 10, wherein the gene is a DMBT1 gene. 13. The method of claim 10, wherein the gene is an Ebnerin gene. 14. DNA comprising the sequence of FIG. 1 or a sequence different therefrom by one or more base pairs, the latter sequence hybridizing with the sequence of FIG. 1 and not being a cDNA. 15. The DNA of claim 14, wherein the DNA is that of FIG. 2. 16. The DNA of claim 14, wherein the DNA is that of Fig. 3A. 17. The DNA of claim 14, wherein the DNA is that of 4B. 18. The DNA of claim 14, wherein the DNA is that of 4C, 1st or 4C, 2nd. 19. Use of the non-human mammal according to one of the claims che 1-8 or the cells according to claim 9 for testing substances, that influence a SCUZ protein or its function or effects wise at least partially replace. 20. Use according to claim 19, wherein the substances for treatment used by carcinomas. 21. Use according to claim 11, wherein the carcinomas are brain tumors, Breast tumors, lung carcinomas, melanomas, pancreas, endometrial, Include esophageal, colon, and gastric cancers.