WO2006085590A1

WO2006085590A1 - TetR EXPRESSING NON-HUMAN TRANSGENIC ANIMAL AND METHOD FOR PRODUCING THE SAME

Info

Publication number: WO2006085590A1
Application number: PCT/JP2006/302265
Authority: WO
Inventors: Yoshitaka Tamai; Yukiko Tsukada; Naomi Murai
Original assignee: Banyu Pharmaceutical Co., Ltd.
Priority date: 2005-02-09
Filing date: 2006-02-09
Publication date: 2006-08-17
Also published as: JP2008104357A

Abstract

[PROBLEMS] To obtain a non-human transgenic mouse that can be used for inducing in vivo expression of a nucleic acid molecule and/or a protein that is not expressed sufficiently when it is induced by a drug substance; and to provide a method for producing the same. [MEANS FOR SOLVING PROBLEMS] With the method for producing a non-human transgenic animal characterized by comprising the steps of producing a TetR gene introduced cell by introducing an expression vector in which TetR gene is located downstream of a promoter into a fertilized egg or an embryonic stem cell; and obtaining a TetR gene introduced non-human transgenic animal by introducing the TetR gene introduced cell into a non-human mammal and the non-human transgenic animal produced by the production method, it becomes possible to obtain a non-human transgenic animal that can be used when the expression of a nucleic acid molecule and/or a protein that is not expressed sufficiently when it is induced by a drug substance is induced in vivo.

Description

Specification

TetR-expressing non-human transgenic animal and method for producing the same

Technical field

[0001] The present invention relates to a TetR-expressing non-human transgenic animal used for inducing expression of a desired gene in vivo.

Background art

[0002] In recent years, along with the progress of genome research, functional analysis of genes is rapidly progressing. Although in vitro experimental systems using recombinant proteins and cultured cells are often used for functional analysis of genes, knowledge obtained from in vitro experiments is not necessarily in vivo. In some cases, it does not reflect the gene function in vivo, and it is very significant for in vivo gene function analysis.

[0003] On the other hand, artificially controlling gene expression is extremely useful for analyzing gene expression and function, and so far, in vitro gene expression control systems have been actively developed. Has been made. Such a gene expression control system can express specific genes by creating knockout animals that are highly useful not only in in vitro experimental systems but also in in vivo experimental systems. Knowledge of the function of the gene is obtained by controlling the amount and analyzing the animal expression system.

[0004] However, in the genetically modified vectors used in conventional transgenic animals and knockout animals, gene expression is arbitrarily controlled, that is, tissue specificity and expression timing are specified to achieve desired tissue and timing. It was not easy to control expression. For example, in a transgenic animal, a gene that is expressed only in the nervous system in the wild type is expressed in all tissues by transgeneicization, and the original function can be explored. There were cases where bad effects such as that which could not be made occurred.

[0005] In order to overcome such harmful effects, for example, Mark Mayford et al. Used calcium-modulo-dependent kinase II (CaMKII) using the forebrain-specific expression motor. We have created transgenic mice that specifically express and have gained knowledge on the function of the molecule (Science, Vol.274, 1678-1). 683, 1996: Non-patent document 1). In this document, a mouse having a promoter that expresses forebrain specifically and a mouse having a tetracycline transactivator system were crossed, and administration of doxycycline to this mouse produced CaMKII. A system that guides the present is used. A similar attempt is also disclosed in MoL Pharmacology, 54, 495-503, 1998 (Non-Patent Document 2).

[0006] Non-Patent Document 1: Science, 274, 1678-1683, 1996

Non-Patent Document 2: Mol. Pharmacology, 54, 495-503, 1998

Disclosure of the invention

Problems to be solved by the invention

[0007] However, in order to induce expression of a nucleic acid having a relatively small molecular weight such as shRNA with a drug, sufficient expression is not observed with a promoter used in Non-Patent Document 1 or 2, and other It was necessary to use an expression system.

[0008] The present invention has been made in view of the above-mentioned problems of the prior art, and is a transgene that can be used for in vivo expression induction of molecules that cannot be expressed sufficiently when induced with drugs. The goal is to get a nick mouse.

Means for solving the problem

[0009] As a result of intensive studies to achieve the above object, the present inventors have used a specific promoter and introduced a sequence that contributes to high-efficiency expression. The present invention was completed by successfully developing a non-human transgenic animal that can be used for inducing expression of molecules and / or proteins at a desired timing.

[0010] That is, the non-human transgenic animal of the present invention is characterized by expressing a TetR protein.

[0011] Here, in the non-human transgenic animal of the present invention, the promoter used for expressing the TetR protein is preferably a CAG promoter. By using such a plug motor, the TetR gene can be expressed more strongly.

[0012] In addition, the TetR protein is expressed in the non-human transgenic animal of the present invention. Insulator sequences are included in the expression vector. By including the insulator sequence, it becomes possible to induce the expression of the transgene with high probability.

[0013] Further, in the non-human transgenic animal of the present invention, an IRES sequence may be contained in an expression vector for expressing the TetR protein. By including the IRES sequence, cells that express the TetR protein at a high level can be selected with high efficiency.

[0014] In addition, in the method for producing a non-human transgenic engineered animal of the present invention, an expression vector in which a TetR gene is arranged downstream of a promoter is introduced into a fertilized egg or embryonic stem cell, and a TetR gene-introduced cell is obtained. And a step of introducing the TetR gene-introduced cell into a non-human mammal to obtain a TetR gene-introduced transgenic animal.

[0015] Here, in the method for producing a non-human transgenic animal of the present invention, as a method for introducing the expression vector into a fertilized egg or embryonic stem cell, an electoral mouth location, a ribofusion, a calcium phosphate coprecipitation method is used. Any one selected from the group consisting of microinjection and viral vector methods is preferably used.

[0016] In the method for producing a non-human transgenic animal of the present invention, it is preferable that the promoter is a CAG promoter. By using such a promoter, the TetR gene can be expressed more strongly.

[0017] Further, in the method for producing a non-human transgenic animal of the present invention, the expression vector may further contain an insulator sequence. By including an insulator sequence, it is possible to induce the expression of the transgene with high probability.

[0018] Further, in the method for producing a non-human transgenic animal of the present invention, the expression vector may contain an IRES sequence. By including the IRES sequence, cells that express TetR protein at a high level can be selected with high efficiency.

[0019] Further, the test nucleic acid Tet-inducible non-human transgenic animal of the present invention is characterized by being produced by crossing the above-mentioned non-human transgenic animal and an animal expressing the test nucleic acid. . Such an animal makes it possible to induce a desired test nucleic acid with tetracycline in vivo, and to analyze the function of the test nucleic acid (target gene).

[0020] In the test nucleic acid Tet-inducible non-human transgenic animal of the present invention, it is preferable that the test nucleic acid is siRNA and / or shRNA. siRNA and shRNA Is a nucleic acid having a relatively small molecular weight, but by using the non-human transgenic animal of the present invention, expression can be induced in vivo by tetracycline.

Here, terms used in the present invention will be described.

The “promoter” in the present invention means a region on DNA that determines the transcription start site of a gene and regulates the frequency of transcription.

[0023] "siRNA (small interfering RNA)" and "shRNA (short hairpin RNA)" are short (about 2 lmer) double-stranded RNAs that cause RNA interference (RNAi). Here, RNA interference is a phenomenon that causes double-stranded RNA force-specific gene sile ndng (gene expression suppression) that is complementary to a certain mRNA and specifically suppresses the expression of the target gene. It is used as a technology.

[0024] The "CAG promoter" is a promoter which has been reported to work strongly in many cells and animals as a tissue non-specific expression promoter.

The invention's effect

[0025] According to the present invention, it is possible to obtain a non-human transgenic animal that can be used when inducing expression of a molecule in vivo when sufficient expression cannot be obtained when induced with a drug. . In other words, it can be used to induce the expression of low molecular weight nucleic acid molecules that cannot be expressed sufficiently when induced with drugs in vivo, or to induce the expression of low expression proteins with high efficiency. Non-human transgenic animals can be obtained.

Brief Description of Drawings

[0026] FIG. 1 is a view showing the expression of TetR protein in each tissue confirmed by Western analysis. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in detail.

[0028] The non-human transgenic animal of the present invention is characterized by expressing TetR protein.

[0029] The expression induction system by the tetracycline gene is a system that utilizes the characteristics of the Tet repressor (TetR protein) and Tet operator (tetO) sequences that act on the transcriptional regulation of the tetracycline-resistant operon of Escherichia coli. According to this system, Tetracycline Nyxixa It is possible to start / stop the expression of the desired gene by induction of iclin and the like (including Tet). For example, in the case of the Tet_off system where transcription is switched in the presence of Tet, the Tet transactivator (tTA) protein forms a complex with Tet, and the complex binds to the TetO tandem sequence. Suppresses the expression of a desired gene downstream of the promoter. On the other hand, in the case of the Tet-on system in which a transcription switch is inserted in the presence of Tet, a reverse Tet transactivator (rtTA) protein in which 4 amino acids of the tTA protein are mutated is used instead of the tTA protein. The rtTA protein represses the transcriptional activity of the Tet-responsive promoter in the absence of Tet and is activated in the presence of Tet.

[0030] Here, a system using tTA or rtTA often uses a pol II promoter transcribed by RNA polymerase II. However, the promoters used in the system have insufficient expression ability to express a relatively small molecular weight nucleic acid (eg, siRNA or shRNA), and sufficient expression cannot be expected. Therefore, in producing the non-human transgenic animal of the present invention, a tetracycline-induced expression system using tTA or rtTA cannot be used.

[0031] Therefore, in view of a strong viewpoint, the present inventors constructed a TetR expression system using a promoter that can be expected to have higher expression or a promoter of Ρο プロモーター system, and desired low molecular nucleic acid and Z or protein. We succeeded in producing transgenic mice that can be used for the expression of.

[0032] The TetR gene according to the present invention may be a mutant having a substitution, deletion, addition or insertion of one or more bases in the TetR gene as long as it encodes a functional protein as a Tet repressor. Yo! In addition, restriction enzyme sites and linkers necessary for DNA recombination can be added to the TetR gene as needed.

[0033] The type of promoter according to the present invention is not particularly limited. For example, the CA G promoter (Niwa, H et al., Gene 107, 193-200 (1991)), tRNA promoter, U6 promoter HI promoter.

[0034] In the expression vector according to the present invention, a TetR gene is arranged downstream of the promoter. Even if the promoter and TetR gene are directly linked, The desired base sequence may be inserted, but it must be functionally linked. here

The term “functionally linked” means that the promoter and the TetR gene are combined so that the expression of the TetR protein is induced with the activation of the transcription of the promoter.

[0035] In addition to the TetR gene and promoter, the expression vector may contain other components necessary for gene expression, detection of the expressed gene, and the like. Examples of such components include an IRES (internal ribosome entry site) lj, a GFP (Green Fluorescent Protein) gene, and an insulator lj.

[0036] Here, the IRES distribution IJ refers to the ribosome binding sequence in the mRNA, and it is incorporated into the expression vector so that one kind of mRNA force has two open reading frames. Translation is possible (J. Virology, 62, p2636_2643 (1988)). In other words, the ribosome translates the target gene (TetR) from the 5 'end of the bicistronic mRNA and also translates the downstream selectable marker and reporter gene from the IRES sequence. As a result, cells that express the target protein at a high level can be selected with high efficiency by the action of a selection marker whose translation efficiency is increased by the IR ES sequence.

[0037] The GFP protein is a fluorescent protein derived from a luminescent jellyfish. For example, a fusion protein of a TetR gene and a GFP gene is constructed and incorporated into an expression vector to express the fusion protein. It emits green fluorescence in the body by irradiating the excitation wavelength. Using this fluorescence as an index, the expression of TetR protein can be detected visually.

[0038] The insulator sequence refers to a DNA segment that has a function of acting on a promoter to enhance or suppress gene expression and a function of avoiding the position effect of the transgene (Cell, 74, p505, (1993)). In transgenic animals, when an exogenous gene is randomly integrated into a chromosome, the expression level of the transgene may differ depending on the location of the integration. In such a case, by sandwiching both sides of the transgene with an insulator sequence, a decrease in the expression level of the transgene due to the position effect is avoided. As a result, transgene expression can be guided with high probability.

[0039] The non-human transgenic animal of the present invention is not particularly limited as long as it is a non-human animal, and examples thereof include rodents such as mice, rats, guinea pigs, and rabbits. Can be mentioned.

[0040] Next, a method for producing a non-human transgenic animal of the present invention will be described.

[0041] First, an expression vector having the TetR gene as described above is constructed by recombinant DNA technology. Enzymatic construction can be carried out by methods well known to those skilled in the art. Enzymes such as restriction enzymes and ligases used for construction may be appropriately selected for the restriction enzyme site to be used. In addition to the above-mentioned components, the expression vector contains a positive selection marker such as a neomycin resistance gene, a hygromycin resistance gene or a puromycin resistance gene in order to select and concentrate the obtained homologous recombinants. In addition, negative selection markers such as diphtheria toxin A gene or simple herpesvirus thymidine kinase gene may be included.

[0042] Next, the constructed expression vector is introduced into an animal to produce a non-human transgenic animal. A powerful non-human transgenic animal can be produced by a method well known to those skilled in the art. For example, an expression vector is introduced into the pronucleus of a fertilized egg, and the resulting cell is returned to the foster oviduct. Method or introducing an expression vector into embryonic stem cells to cause homologous recombination, selecting the obtained homologous recombinants, and returning the homologous recombinants into the blastocyst space to form a chimeric embryo And then transplanting it into the womb of a deceased parent to produce a chimeric animal and crossing this chimeric animal with a wild type animal to produce a heterozygous non-human transgenic animal, A non-human transgeneic animal that is a heterozygote obtained in this way can be bred with each other to further produce a nonhuman transgeneic.

[0043] In addition, a method for introducing an expression vector into a fertilized egg or embryonic hepatocyte can also be carried out by a method well known to those skilled in the art. For example, electopore position, lipofuxion, calcium phosphate coprecipitation method, microinjection, and viral vector The method by is mentioned. Examples of the method using the virus vector include methods using SV_40, adenovirus, herpesvirus, adeno-associated virus (AAV), vaccinia virus, ushipapilloma virus, and retrovirus as virus vectors. It is preferable to use retrowinoles (for example, lentivirus) as a viral vector.

[0044] In the thus-produced non-human transgenic animal, the TetR protein is expressed in the tissue based on the tissue specificity of the expression of the promoter used. Therefore, power The expression of the test nucleic acid can be induced by Tet administration by crossing the non-human transgenic animal with the non-human transgenic animal into which the test nucleic acid (transgene) has been introduced. Human transgenic animals can be generated. In this case, even if the test nucleic acid is a nucleic acid having a relatively small molecular weight (siRNA or shRNA) or a low molecular weight nucleic acid that is difficult to express in a normal expression system, the expression can be induced. In addition, in a protein with low expression efficiency, it becomes possible to induce expression of the target protein with high efficiency.

[0045] Here, "a non-human transgenic animal into which a test nucleic acid (transgene) has been introduced" is an expression vector that contains a TetO sequence that is a binding sequence of TetR protein in addition to the test nucleic acid (transgene). It is necessary to have one. That is, it is necessary to have an expression vector in which a test nucleic acid (transgene) is arranged downstream of the TetO sequence that is a binding sequence of the TetR protein. By crossing with the non-human transgenic animal of the present invention, the test nucleic acid can be expressed in a Tet-inducible manner. Furthermore, an expression vector for a test nucleic acid (transgene) transgene is further introduced into an egg (TetR + egg) or tissue (TetR + tissue) isolated from the non-human transgenic animal of the present invention. By returning to the foster parent and obtaining a chimeric animal, it becomes possible to obtain a model animal capable of expressing the test nucleic acid in a Tet-inducible manner. The non-human transgenic animal capable of expressing the test nucleic acid in a Tet-inducible manner can be used for functional analysis of the test nucleic acid (target gene), and the test nucleic acid (target gene) can be used. It is useful as a model animal for diseases involving offspring. Example

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

(Construction of TetR expression vector and generation of transgenic mice)

[0047] 1. Preparation of pGlobin-TetR-IRES-EGFP plasmid

Using the pcDNA6TR plasmid (Invitrogen) as a template, the following primers were used, and the regions of Rabitt beta globin intron II and TetR coding sequence were amplified by PCR. The primer contains a Pstl cleavage site and an Xhoi cleavage site. G (SEQ ID NO: 1)

XhoI-globin-PCR: CTCGAGGTGA GTTTGGGGAC CCTTGATTGT TC (SEQ ID NO: 2)

[0048] Next, the amplified 1.3 kb fragment was cloned as a blunt end into the EcoRV site of pBluescript SK (-). From the cloned plasmid, fragments of the Rabitt beta globin intron II and TetR coding sequences were excised at the Pstl and Xhol cleavage sites.

[0049] On the other hand, pIRES-EGFP plasmid (Clontech) was cleaved at the Pstl cleavage site and the Xhol cleavage site. By ligating both, the pGlobin-TetR-IRES-EGFP plasmid was cloned.

[0050] 2. Preparation of pSK / CAGGS

Cut out the CAG promoter from pCAGGS (Chemical and Serological Therapy Laboratory) with Sspl and Hindlll, and after blunt-end treatment with the prepared Smal / Hindlll-cut pBluescript SK (-) plasmid. Ligation was performed to clone the pSK / CAGGS plasmid.

[0051] 3. Preparation of pSK / CAG-TetR-IRES-EGFP plasmid

pGlobin-TetR—IRES-EGFP plasmid strength, excise the Globin-TetR-IRES-EGFP fragment with Xhol and Notl, and the EcoRI-cut pSK / CAGGS plasmid prepared in advance and ligated after blunt end treatment and pSK The / CAG-TetR-IRES-EGFP plasmid was cloned.

[0052] 4. Preparation of pSKM / Ins-CAG-TetR-IRES-EGFP plasmid

In pSK / CAG-TetR-IRES-EGFP, a 1.2 Kb insulator sequence at the No tl cleavage site located 5 'upstream of the CAG promoter and the Clal cleavage site located 3' downstream of the polyA signal sequence, respectively. Were cloned in tandem. That is, a pSKM / 1 ns-CAG-TetR-IRES-EGFP plasmid in which an CAG-TetR_IRES-EGFP expression cassette was sandwiched between insulator sequences was prepared. As the insulator sequence, the sequence reported in Cell 74, pp505-514 was used.

[0053] 5. Preparation of pCAG-TetR plasmid Using the pcDNA6TR plasmid (Invitrogen) as a template and the following primers, the TetR coding sequence region was amplified by PCR.

(Column number 4)

[0054] After the amplified fragment of about 1.3 kb was cloned into pBluescript SK (-), Eco

A fragment of the TetR coding sequence was excised with RV.

[0055] On the other hand, the pCAGGS plasmid (Chemical and Serological Therapy Laboratory) was cleaved with EcoRI, and blunt-ended with a fragment of the TetR coding sequence after the blunt end treatment to clone the pCAG-TetR plasmid.

[0056] 6. Preparation of pSKM / Ins-CAG-TetR plasmid

On the pCAG-TetR plasmid, two 1.2 Kb insulator sequences were cloned in tandem at the Notl cleavage site located 5 'upstream of the CAG promoter and the Clal cleavage site located 3' downstream of the polyA signal sequence, respectively. That is, a pSKM / Ins-CAG-TetR plasmid was prepared in which the CAG_TetR-IRES-EGFP expression cassette was sandwiched between insulator sequences. As the insulator sequence, the sequence reported in Cell 74, pp505-514 was used.

[0057] 7. Production of transgenic mice

In order not to contain the cloning vector sequence as much as possible from each of the four types of plasmids cloned as described above, the insert and the vector were separated by electrophoresis on agarose gel after digestion with restriction enzymes as follows.

pCAG-TetR (SspI / Pstl)

pSKM / Ins-CAG-TetR (Clal / Clal)

pSK / CAG— TetR-IRES— EGFP (Notl / Clal)

pSKM / Ins-CAG-TetR- IRES-EGFP (Clal / Clal) ₀

[0058] Next, only the gel force insert DNA fragment was excised, the DNA fragment was purified using a DNA fragment purification kit (Qiagen), and dissolved in TE buffer. The DNA solution thus obtained is filled into a glass capillary and microinjected into the female pronucleus of a fertilized egg of a mouse. Injected. Furthermore, after the fertilized egg was transplanted into the oviduct of a foster mother of pseudopregnancy, a transgenic mouse was obtained by spontaneous delivery.

(Construction of TetR expression vector and creation of Knock in mouse by homologous recombination)

[0059] 1. Preparation of Knock in vector

Using the ROSA26 locus (Pro Natl. Acad. Sci. USA, 94, 3789-3794, 1997) that is ubiquitously expressed during embryonic development, a TetR Knock in vector was prepared.

[0060] Specifically, an approximately 4.3 kb nucleic acid fragment excised from the ROSA26 locus with Sadl and Xbal was inserted into the SacII and Xbal cleavage sites of the pGK-DT-A-bpA vector. Next, PGK-neo-bpA / CAG-TetR-IRES-E GFP was inserted into the Xbal cleavage site located about 0.9 kb from the SacII cleavage site to obtain a TetR-EGFP Knock in vector.

[0061] 2. Preparation of Knock in Mouse

By introducing the prepared Knock in vector into embryonic stem cells using electoporation and culturing in the presence of G418, clones in which homologous recombination occurred between the CAG-TetR-IRES-EGFP sequence and the ROSA26 locus Obtained. In addition, it was confirmed by Southern blotting that the Knock in vector was introduced into the target gene locus.

[0062] Next, embryonic stem cells in which homologous recombination occurred were introduced into blastocysts by microinjection to form a chimeric embryo, which was then transplanted into a temporary parent uterus to obtain a chimeric mouse. When PCR was performed using the genomic DNA extracted from the tail of the chimeric mouse obtained as a saddle type, it was confirmed that the TetR gene was introduced. In addition, TetR protein expression in each tissue was confirmed by Western analysis (Fig. 1) Industrial applicability

[0063] According to the present invention, it is possible to obtain a transgenic animal that can be used for inducing expression of a nucleic acid molecule and / or protein in vivo when sufficient expression is not obtained when induced with a drug. It becomes possible. Therefore, control the expression of the desired gene by expressing siRNA and / or shRNA against the gene of interest using a powerful non-human transgenic animal or by expressing a protein that is the translation product of the gene. And the in vivo function of the gene can be analyzed.

Claims

The scope of the claims

[I] A non-human transgenic animal characterized by expressing TetR protein.

[2] The non-human transgenic animal according to claim 1, wherein the promoter used for expressing the TetR protein is a CAG promoter.

[3] The non-human transgenic animal according to claim 1 or 2, wherein the expression vector for expressing the TetR protein contains an insulator sequence.

[4] The non-human transgenic animal according to any one of claims 1 to 3, wherein the expression vector for expressing the TetR protein contains an IRES sequence.

[5] A step of introducing a TetR gene-introduced cell by introducing an expression vector in which the TetR gene is arranged downstream of the promoter into a fertilized egg or embryonic stem cell;

A method for producing a non-human transgenic animal, comprising the step of introducing the TetR gene-introduced cell into a non-human mammal to obtain a TetR gene-derived transgenic animal.

[6] As a method for introducing the expression vector into a fertilized egg or embryonic stem cell, any one selected from the group consisting of a method using an electoral mouth position, a rib function, a calcium phosphate coprecipitation method, a microindication and a virus vector 6. The method for producing a non-human transgenic animal according to claim 5, wherein one is used.

[7] The method for producing a non-human transgenic animal according to [5] or [6], wherein the promoter is a CAG promoter.

[8] The method for producing a non-human transgenic animal according to any one of [5] to [7], wherein the expression vector contains an insulator sequence.

[9] The method for producing a non-human transgenic animal according to any one of [5] to [8], wherein the expression vector contains an IRES sequence.

[10] A test nucleic acid produced by crossing the non-human transgenic animal according to any one of claims 1 to 4 with an animal that expresses the test nucleic acid. Human transgenic animal.

[I I] The non-human transgenic animal according to claim 10, wherein the test nucleic acid is siRNA or shRNA.