JP5173218B2 - Egg white specific substance production by ovalbumin promoter - Google Patents

Description

The present invention relates to a method for producing a transgenic bird that expresses a foreign target protein specifically in the fallopian tube, and a protein pharmaceutical production method that accumulates and recovers a foreign target protein in eggs laid by the transgenic bird and offspring.

It has been industrially produced by producing protein drugs in microorganisms and eukaryotic cells, which can be extracted only from a biological sample by genetic recombination technology and difficult to produce by chemical synthesis. For the production of useful proteins using recombinant technology, prokaryotic organisms such as E. coli, eukaryotic organisms such as yeast, and systems that use cultured cells of mammalian cells and insect cells have been successful. ing.
However, as the protein dynamics in the cell become clear in detail as a result of remarkable research in recent years, specific and functional proteins that focus on post-translational modification, folding, and higher-order structures as protein production methods desired as pharmaceuticals The technology to produce is desired. The production method of highly functional protein preparations is mainly based on mammalian cells, but this production method has disadvantages such as high cost, poor productivity, and risk of infection due to virus contamination. To do.

Therefore, attention was focused on a method for producing protein pharmaceuticals using transgenic animals. The development of transgenic animals has been reported all over the world, and transgenic goats and sheep in which useful proteins for cytokines and blood products are accumulated in milk are already in practical use. The merit of protein production by these transgenic animals is that high-functional proteins having post-translational modification, folding, and higher-order structures similar to the production system in mammalian cells can be produced in large quantities at low cost. Large-sized mammals are used in protein drug production by transgenic animals, but these techniques for producing milk from large mammals such as cows, sheep, and goats have matured into a large breeding space and established a production line. There is a drawback that takes a long time to do.
So we focused on chickens. A technique for producing useful proteins in transgenic chickens has been developed (Patent Document 1), and the technique of recovering the target protein from this egg does not require a large breeding space, and the egg-laying time is as short as 6 months. The point that safety is confirmed from experience is useful. Conventionally, a systemic expression promoter has been used as a production method for this transgenic chicken, but it is not suitable for the production of proteins that are toxic to chickens. Therefore, a technique for expressing and accumulating a target protein specifically for chicken eggs is required.

In order to accumulate the target protein in an egg-specific manner, a promoter that is specifically expressed in an oviduct cell that produces egg white may be applied. Patent Document 2 discloses a method for producing a transgenic bird using an egg white promoter, but in this technique of microinjecting a retrovirus into an X-stage fertilized egg, the target sequence can be incorporated into the host, but the mechanism is unknown. It was found that expression was suppressed by gene silencing (Patent Document 1), and it was impossible to express specifically in the fallopian tube cells.
Further, Patent Document 3 discloses a method for producing a transgenic chicken using a chicken ovalbumin promoter, but the productivity is low and it is not suitable for practical use. In Patent Document 3, although the expression level was very low, tissue-specific expression could be realized. Therefore, in order to improve the expression level, a solution for introducing a gene sequence that enhances transcriptional activity can be considered. However, due to the characteristics of viral vectors that cannot introduce long gene sequences, it is impossible to introduce sequences other than the long-chain ovalbumin promoter and the target gene.
International Publication No. 2004/016081 Pamphlet Special table 2000-512149 gazette JP 2004-236626 A

Although a production method using eggs from transgenic chickens has been proposed as a method for producing highly functional proteins for pharmaceuticals, expression is lethal because the expression level of the systemic expression promoter is high but the target protein is toxic to the host. There are problems. Thus, as a universal method for producing highly functional proteins in eggs, there is a need for a technique that enables expression of foreign target protein genes specific to the fallopian tubes using a promoter sequence that functions specifically in the fallopian tubes. . Japanese Patent Application Laid-Open No. 2004-236626 discloses a method for producing a transgenic chicken using a retroviral vector incorporating a chicken ovalbumin promoter. However, in this method, an expression level at a practical level could not be obtained. Oviduct-specific expression and practical protein production methods are required.

The present inventors cut the ovalbumin promoter to a chain length exhibiting tissue-specific transcriptional activity and enhanced transcriptional activity by inserting a transcriptional activation sequence, so that the target protein can be expressed specifically in the oviduct cell. By constructing a strong expression vector system and applying it to transgenic birds, we established a method for producing egg-specific foreign target proteins using transgenic chickens.
The present invention highly expresses a foreign target protein specifically in avian oviduct cells in a transgenic bird, highly accumulates the foreign target protein specifically in an egg laid by the bird, and recovers it. This is a method for producing a foreign protein of interest.
High production of a foreign target protein in avian oviduct cells can be achieved by using the ovalbumin promoter to achieve oviduct-specific expression and enhancing its transcriptional activity. The ovalbumin promoter disclosed in Japanese Patent Application Laid-Open No. 2004-236626 has a long sequence, and in view of the length used for a retroviral vector, other transcription activation sequences cannot be inserted.
Therefore, truncating the ovalbumin promoter sequence, obtaining a sequence as short as possible that can be expressed in the fallopian tube, and incorporating a transcription enhancing factor to produce a retroviral vector that enables expression of the target protein specific to the fallopian tube, An attempt was made to produce transgenic birds using the vector. As the ovalbumin promoter DNA sequence, the transcription start point of chicken ovalbumin gene was 0 (reference), and about 2.3 kbp from −3860 to −1568 was used upstream (SEQ ID NO: 2). A retroviral vector that enhances the transcriptional activity of the ovalbumin promoter was prepared by incorporating tetracycline-responsive herpesvirus-derived VP16 factor as a transcriptional activator, and a transgenic chicken that was highly expressed in the oviduct was obtained. Successfully produced.

Therefore, the present invention provides the following.
(1)
A promoter for gene expression of the ovalbumin promoter,
a) having all or part of the hormone-dependent DNase I supersensitive site III;
b) does not contain hormone-dependent DNase I supersensitive site I and / or hormone-dependent DNase I supersensitive site II,
c) functioning specifically in the oviduct in birds, not exceeding 3 kbp and exceeding 1.0 kbp,
d) including the TATA box,
A promoter consisting of a partial region.
(2)
The promoter according to (1), wherein the ovalbumin promoter is an avian ovalbumin promoter.
(3)
The promoter according to (2), wherein the avian ovalbumin promoter is a chicken ovalbumin promoter.
(4)
The promoter according to (3), wherein the chicken ovalbumin promoter is the chicken ovalbumin promoter described in SEQ ID NO: 1 in Sequence Listing.
(5)
A promoter consisting of the base sequence shown in SEQ ID NO: 2 or consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 2, and having an activity that functions specifically in the oviduct in birds.
(6)
A promoter consisting of the base sequence shown in SEQ ID NO: 3 or a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 3, and having an activity that functions in an oviduct-specific manner in birds.
(7)
A viral vector having the promoter according to any one of (1) to (6).
(8)
The viral vector according to (7), which is a retroviral vector.
(9)
The virus vector according to (8), further comprising a foreign target protein gene incorporated therein.
(10)
The promoter according to any one of (1) to (6) induces an oviduct-specific expression of an activator protein, and induces expression of a foreign target protein gene in cis and / or trans by the activator protein. The viral vector according to claim 9.
(11)
The viral vector according to (10), further comprising an enhancer, a signal sequence, and a post-transcriptional regulator.
(12)
(7) Transgenic birds produced by the virus vector according to any one of (11).
(13)
(12) The transgenic bird obtained by microinjecting a virus vector into the embryonic heart at the stage of development of a fertilized bird bird and allowing it to hatch.
(14)
A progeny transgenic bird obtained by crossing the transgenic bird according to (12) or (13) with another bird.
(15)
The progeny transgenic bird according to (14), wherein the exogenous target protein is predominantly expressed in oviduct cells of a female individual.
(16)
(15) An egg containing a foreign protein of interest in egg white or egg yolk laid by a progeny transgenic bird.
(17)
A method for producing a foreign target protein by the transgenic bird according to (12) or (13) or the progeny transgenic bird according to (14) or (15).
(18)
The method for producing a foreign target protein according to (17), wherein the foreign target protein is an antibody, cytokine, insulin, hormone, bioactive protein, or a fusion protein comprising these.
(19)
The method for producing a foreign target protein according to (17) or (18), wherein the transgenic bird is a transgenic chicken.

The present invention is described in detail below.
The method for producing a foreign target protein of the present invention comprises using an ovalbumin promoter truncated to a chain length exhibiting tissue-specific transcriptional activity, which comprises expressing the foreign target protein in egg-specific high expression in transgenic birds. Features.
The truncated ovalbumin promoter has all or part of the hormone-dependent DNaseI supersensitive site III, does not contain the hormone-dependent DNaseI supersensitive site I or the hormone-dependent DNaseI supersensitive site II, and is a fallopian tube in birds Any partial region (fragment) that is 3 kbp or less and exceeds 1.0 kbp that specifically functions and includes a TATA box is included in the technical scope of the present invention.
The ovalbumin promoter is preferably an avian ovalbumin promoter, more preferably a chicken ovalbumin promoter. Sequence number 1 of the sequence listing shows the sequence of the chicken ovalbumin promoter.
The partial region of the truncated ovalbumin promoter of the present invention is 3 kbp or less, and preferably 1.4 kbp or less. This truncated promoter is preferably 1.1 kbp or more.

Examples of the truncated ovalbumin promoter of the present invention include those described in SEQ ID NO: 2 or 3 in Sequence Listing.
In addition, promoters of the present invention include promoters comprising a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 and having an activity that functions specifically in the oviduct in birds. . More preferred is a promoter of a base sequence having 90% or more identity, more preferably 95% or more identity, particularly preferably 99% or more identity.
Here, “identity (%)” means that the two polynucleotides to be compared are optimally aligned and the nucleobases (eg, A, T, C, G, U, or I) match in both sequences. The number of positions is divided by the total number of comparison bases, and the result is expressed by a value obtained by multiplying by 100.
Sequence identity can be calculated using, for example, the following sequence analysis tools: GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group 37, 575 ScienceDenM Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Center, Hinxton Hall, Cambridge, CB10 1RQ, England, and the ExPASyWr ty Hospital and University of Geneva, Geneva, Switzerland).

Transgenic birds used in the method for producing a foreign target protein of the present invention can be obtained by introducing a viral vector containing the above truncated ovalbumin promoter into birds.
The viral vector is preferably a replication-defective viral vector. The replication-defective virus vector is not particularly limited as long as it is a vector capable of introducing a target gene sequence into a host bird, such as a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a box virus, or a herpes virus.
In the present invention, the viral vector is preferably one in which a foreign target protein gene is further incorporated in order to cause transgenic birds to produce a foreign target protein. In Examples described later, DsRed was used as a foreign target protein gene.
As the foreign target protein, antibodies, cytokines, insulin, hormones, physiologically active proteins or fusion proteins comprising these are preferred.

The sequence to be incorporated into the replication-defective virus vector is not particularly limited as long as it is a vector construct that contains a truncated ovalbumin promoter and can efficiently accumulate a foreign target protein in the eggs of transgenic birds. A vector construct incorporating a gene sequence encoding an activator and a cis or transactivator system that strongly expresses a downstream target gene by the transcription activator is preferred.
As a more preferred system, a system using a tetracycline responsive element and a herpes virus-derived VP16 transactivator is preferable, but the system is not limited to this, and a galactose responsive element or the like can also be used.

In addition, this viral vector may incorporate a DNA sequence for the purpose of efficiently expressing a foreign transgene, such as a post-transcriptional regulatory factor, an enhancer sequence, and a signal sequence.

Transgenic birds (G0 transgenic chimeric birds) can be produced by introducing the above-described viral vectors into host birds.
In the present invention, the foreign target protein gene is introduced into a virus vector different from the virus vector into which the truncated ovalbumin promoter has been introduced, and two kinds of virus vectors are introduced into the host birds to produce G0 transgenic chimeric birds. You can also
Examples of methods for producing G0 transgenic chimeric birds used in the present invention include the methods implemented by the present inventors (Japanese Patent Laid-Open Nos. 2002-176880, 2004-236626, and International Publication). No. 2004/016081 pamphlet), but is not limited thereto, but can also be applied to a method for producing transgenic birds by a non-viral method disclosed in JP-T-2005-535331 and JP-A-2006-262875. It is not limited to. Any known technique for microinjecting a replication-defective viral vector into the embryo, heart or blood vessel of an avian fertilized egg is preferable because of its high progeny acquisition efficiency. In the present invention, the virus vector is preferably microinjected and hatched into the embryonic heart at the developmental stage of the avian fertilized egg.

Chickens, quails, turkeys, ducks, etc. are used as host birds, but chickens and quails are easy to obtain and prolific as spawning species, and are safe due to many years of breeding experience. It is preferable in that the property is recognized. A chicken is more preferable.

Here, the G0 transgenic chimera bird refers to a first-generation bird produced by the transgenic production method of the present invention, and refers to a bird in which a gene is introduced into its somatic cell in a mosaic chimera form.

The method for selecting the prepared G0 transgenic chimeric bird is not particularly limited, but it is possible to confirm whether the target sequence has been introduced by, for example, PCR or Southern blotting after genome extraction. A female individual of this G0 is grown until it lays eggs using any known animal husbandry technique, and it is confirmed whether the target protein is accumulated in the eggs. The method used for confirmation is not particularly limited. For example, immunochemical quantification and measurement using a specific antibody, or in vitro or in vivo activity measurement using cultured cells or animal tests may be performed. .

By mating the G0 transgenic chimeric birds, progeny can be obtained as fully transgenic individuals having the transgene in all somatic cells. The mating method is not particularly limited, such as natural mating or artificial mating. In the mating, the wild-type bird may be applied to the G0 transgenic chimeric bird, or the G0 transgenic animals may be crossed together. This progeny completely transgenic bird is designated as G1, G2, G3, etc.
In the method for producing a foreign target protein of the present invention, it is preferable to use a transgenic bird that preferentially expresses the foreign target protein in female oviduct cells.
The eggs of the completely transgenic birds after G0 or G1 are collected, and the target protein is collected from the egg white or egg yolk. The recovery method is not particularly limited, and any known purification or separation method such as filtration, affinity chromatography, cation, or anion exchange chromatography can be used.
The target protein thus obtained can be used for pharmaceutical applications.

According to the present invention, a foreign target protein can be highly expressed specifically in the fallopian tube in transgenic birds. Using this technology, it becomes possible to mass-produce highly functional proteins for pharmaceuticals in transgenic birds including proteins that are toxic to the host at low cost.

Hereinafter, although the preferable form which implements this invention is explained in full detail, the essence of this invention should not be unduly limited by the content of description.
(Example 1) Acquisition of a truncated ovalbumin promoter sequence
1-1 Construction of pBlueOVA (d) The vector pLGOG described in Japanese Patent Application Laid-Open No. 2004-236626 is treated with restriction enzymes EcoRV (TAKARA) and BamHI (TAKARA), and the purpose is 1% agarose electrophoresis. A 2.4 kb fragment was separated, cut out from the gel, and purified by Mag Extractor-PCR & Gel Clean up- (TOYOBO). This fragment was used as an insert. Further, pBluescript KS- (Novagen) was treated with restriction enzymes EcoRV and BamHI, cut out from the gel, and purified by Mag Extractor-PCR & Gel Cleanup- as a vector.
After confirming the concentration and degree of purification of each purified DNA fragment by 1% agarose electrophoresis, ligation and transformation into E. coli DH5α (TAKARA) were performed. In addition, Ligation reaction was performed according to the protocol using LigaFast Rapid DNA Ligation System (trademark, Promega). In the transformation, first, the competent cell DH5α frozen in liquid nitrogen was thawed, 10 μl of the solution after the above ligation reaction was added to the solution, and the mixture was allowed to stand on ice for 30 minutes. Then, heat shock was performed at 42 ° C. for 45 seconds and ice-cooled for 5 minutes. 1 ml of LB medium was added to this cell suspension, shaken at 37 ° C. for 1 hour, seeded on an LB plate containing 0.1 mg / ml ampicillin, and cultured at 37 ° C. for 14 hours. Thereafter, the target plasmid was obtained from the colonies cultured on the LB plate by the alkali method.

1-2 Construction of pETOVA (BglII-XhoI) First, a genome DNA was prepared from a virus-producing packaging cell pLGOG established by the method described in Examples of JP-A-2004 / 236626 by Mag Extractor-genome- (TOYOBO). Extracted. The packaging cell pLGOG is obtained by using GP293 having gag and pol genes on the chromosome as a virus producer cell and introducing the virus vector pLGOG (GFP, Ovalbumin promoter, hGH (cDNA)), which is the target gene, into the GP293 cell. It is a cell line that is stably expressed and obtained in (1).
PCR reaction was performed using the following primers with the genomic DNA obtained by the above method as a template.
5 'primer (SEQ ID NO: 4)
3 'primer (SEQ ID NO: 5)
Moreover, KOD-Plus-DNA Polymerase (TOYOBO) was used as the enzyme for the PCR reaction, and the conditions were as follows.
(1) 94 ° C. for 15 seconds, (2) 58 ° C. for 30 seconds, (3) 68 ° C. for 15 seconds, (1) to (3) for 35 cycles.
The solution after the PCR reaction was subjected to phenol chloroform extraction and ethanol precipitation, and further subjected to a restriction enzyme reaction with BglII (TAKARA) and XhoI (TAKARA). Thereafter, a target fragment of about 220 bp was separated by 1% agarose electrophoresis, cut out from the gel, and purified by Mag Extractor-PCR & Gel Clean up-.
Similarly, pET Blue2 (Novagen) was subjected to a restriction enzyme reaction with BglII and XhoI, and then the target fragment was separated by 1% agarose electrophoresis, cut out from the gel, and purified by Mag Extractor-PCR & Gel Cleanup-. After confirming the concentration and purity of each purified DNA fragment by 1% agarose electrophoresis, ligation and transformation into E. coli DH5α were performed. The insert used was an about 220 bp fragment prepared after amplification by PCR, and the vector used was the above pET Blue2 fragment. Thereafter, the target plasmid was obtained by the alkali method.

1-3 Construction of pBlueOVA (d) ΔBglII pETOVA (BglII-XhoI) and pBlue OVA (d) were treated with restriction enzymes BglII and XhoI, fractionated and fragmented by 1% agarose electrophoresis, Mag Extractor-PCR & Gel Purification by Clean up- was performed. Ligation and transformation were performed using a fragment of about 220 bp obtained from pETOVA (BglII-XhoI) as an insert and pBlue OVA (d) treated with a restriction enzyme as a vector. Thereafter, the target plasmid was obtained by the alkali method.

1-4 Construction of pBlueOVA Treat each of pBlueOVA (d) and pBlueOVA (d) ΔBglII with BglII, fractionate the target fragment by 1% agarose electrophoresis, cut out, and purify by Mag Extractor-PCR & Gel Cleanup- It was. Ligation and transformation were performed using the 1.2 kb fragment obtained from pBlueOVA (d) as an insert and the fragment obtained from pBlueOVA (d) ΔBglII as a vector. Thereafter, the target plasmid was obtained by the alkali method.

1-5 Construction of pETOVA pBlueOVA and pET Blue2 were treated with restriction enzymes NotI (TAKARA) and XhoI, respectively, fractionated and excised by 1% agarose electrophoresis, and purified by Mag Extractor-PCR & Gel Cleanup- . Ligation and transformation were performed using the fragment obtained from pBlue OVA (complete) as an insert and the fragment obtained from pET Blue2 as a vector. Thereafter, the target plasmid was obtained by the alkali method.

1-6 Confirmation of sequence The nucleotide sequence of the cloned 2.3 kb ovalbumin sequence was confirmed using the following primers. The template was used as pBlueOVA.
OVA-1 (5 ′ primer) (SEQ ID NO: 6)
OVA-2 (5 ′ primer) (SEQ ID NO: 7)
OVA-3 (5 ′ primer) (SEQ ID NO: 8)
OVA-4 (5 ′ primer) (SEQ ID NO: 9)
OVA-5 (5 ′ primer) (SEQ ID NO: 10)
OVA-6 (5 ′ primer) (SEQ ID NO: 11)
OVA-7 (3 ′ primer) (SEQ ID NO: 12)

A truncated ovalbumin promoter sequence (SEQ ID NO: 2) was obtained as described above.

(Example 2) Construction of pQeGOVATRETTAW autoactive virus vector
2-1 Construction of pMSCVeGTREGITTAW
2-1-1 Construction of pMSCVeGTREG The EGFP sequence was amplified by PCR from pIRES2-EGFP (Clontech), treated with restriction enzymes EcoRI and BamHI, and then the vector incorporated into the EcoRI and BamHI sites of pBluescript KS- (Novagen) was added to pBlue. / EG. A fragment obtained by treating pBlue / eG with restriction enzymes EcoRI and XhoI was used as an insert, and a vector integrated into the EcoRI and XhoI sites of pMSCVneo (Clontech) was used as pMSCV / eG. A fragment obtained by treating the TRE sequence with restriction enzymes XhoI and HindIII from pTRE2 (Clontech) was used as an insert, and a vector integrated into the XhoI and HindIII sites of pMSCV / eG was called pMSCV / eGTRE.
pCEP4-hGH (cDNA) in which hGH (cDNA) cloned by JP-A-2004-236626 is incorporated into the restriction enzyme BamHI and MluI sites of pCEP4 (invitrogen) is used as an insert, and a fragment excised with BamHI and MluI is used as an insert. PMSCV / eGTRE was subjected to restriction enzyme treatment with BamHI and MluI, the target band was separated by agarose gel electrophoresis (1%), cut out, and collected by Mag Extractor-PCR & Gel Clean up-. After the recovered DNA concentration was confirmed by agarose electrophoresis (1%), a plasmid obtained by ligation, transformation, and alkali method was designated as pMSCVeGTREG.

2-1-2 Acquisition of cellular IRES (5'-side restriction enzyme MluI site, 3'-side restriction enzyme EcoRI site) Restriction enzyme MluI site on the 5 'side of the sequence of cellular IRES and restriction on the 3' side 5 ′ DNA sequence with the enzyme EcoRI site (SEQ ID NO: 13), 3 ′ DNA sequence with restriction enzyme MluI site on the 3 ′ side of the cellular IRES sequence and 5 ′ side with the restriction enzyme EcoRI site (arrangement) Sequence listing SEQ ID NO: 14) was defined as a synthetic DNA sequence. However, when this synthetic DNA is ligated to the normal 5′-side restriction enzyme MluI site and 3′-side restriction enzyme EcoRI site, the base sequence is not cut by the restriction enzyme EcoRI.

2-1-3 Construction of pET-IRES (r) pET Blue2 (Novagen) as a vector was treated with restriction enzymes MluI and restriction enzyme EcoRI, and the target band was obtained by agarose gel electrophoresis (1%). It isolate | separated, cut out, and collect | recovered by Mag Extractor-PCR & Gel Clean up-. After confirming the recovered DNA concentration by agarose electrophoresis (1%), the plasmid obtained by ligation, transformation, and alkali method using the vector DNA and the cellular IRES synthesized by 2-1-2 as an insert was used as pET- IRES (r).

2-1-4 Construction of pBlue-tTA IRES was amplified by PCR from pIRES2-EGFP (Clontech), and the fragments treated with restriction enzymes BamHI and PstI were used as inserts and assembled at the BamHI and PstI sites of pBluescript KS- (Novagen). The inserted vector was designated as pBlue / I.
pTET-OFF (Clontech) was digested with BamHI, blunted with Klenow (TAKARA), treated with EcoRI as an insert, and the vector integrated into the EcoRI and EcoRv sites of pBlue / I was designated as pBlue / ItTA. Treat pBlue / ItTA with restriction enzymes EcoRI and HindIII as inserts, pBluescript KS- (Novagen) as restriction vectors with restriction enzymes EcoRI and HindIII, respectively, and separate target bands by agarose gel electrophoresis (1%). Then, it was cut out and recovered by Mag Extractor-PCR & Gel Clean up-. After confirming the recovered DNA concentration by agarose electrophoresis (1%), the plasmid obtained by ligation, transformation, and alkali method was designated as pBlue-tTA.

2-1-5 Construction of pET-ItTA pBlue-tTA was treated with restriction enzymes BamHI and PvuII as inserts, and pET-IRES (r) was treated with restriction enzymes BamHI and EcoRV as vectors, and agarose gel electricity The target band was separated by electrophoresis (1%), excised, and collected by Mag Extractor-PCR & Gel Clean up-. After confirming the recovered DNA concentration by agarose electrophoresis (1%), a plasmid obtained by ligation, transformation, and alkali method was designated as pET-ItTA.

2-1-6 Construction of pMSCVeGTREGItTA pET-ItTA as an insert was treated with restriction enzymes MluI and HindIII, and pMSCVGTREG as a vector was treated with restriction enzymes MluI and HindIII, followed by agarose gel electrophoresis (1%). Bands were separated, excised and recovered with Mag Extractor-PCR & Gel Clean up-. After the recovered DNA concentration was confirmed by agarose electrophoresis (1%), the plasmid obtained by ligation, transformation, and alkali method was designated as pMSCVeGTREGITTA.

2-1-7 Construction of pMSCVeGTREGItTAW As an insert, pBlue / WPRE described in JP-A-2006 / 271266 is treated with restriction enzyme ClaI and pMSCVGTREGREGItTA is treated with a restriction enzyme ClaI as a vector, followed by agarose gel electrophoresis (1 %), The target band was separated, cut out, and recovered by Mag Extractor-PCR & Gel Clean up-. After the recovered DNA concentration was confirmed by agarose electrophoresis (1%), the plasmid obtained by ligation, transformation, and alkali method was designated as pMSCVeGTREGITTAW.

2-2 Construction of pMSCVeGOVAREGREGItTAW The pETOVA constructed in Example 1 was restricted with SalI (TAKARA) and XhoI, and pMSCVeGTREGItTAW was restricted with XhoI, and the target fragment was fractionated by 1% agarose electrophoresis. It cut out and refine | purified by Mag Extractor-PCR & Gel Clean up-. Ligation and transformation were performed using the approximately 2.3 kb fragment obtained from pETOVA as an insert, and using pMSCVeGTREGITTAW treated with a restriction enzyme as a vector. Thereafter, the target plasmid was obtained by the alkali method.

2-3 Construction of pETtTAW The viral vector plasmids pMSCVeGOVATREGITTAW and pET Blue2 constructed in 2-2 were each treated with EcoRI, and fractionated and excised from the target fragment by 1% agarose electrophoresis, Mag Extractor-PCR & Gel Clean up- Purification was performed. Ligation and transformation were performed using a fragment of about 1.7 kb obtained from pMSCVeGOVATREGITTAW as an insert and pET Blue2 treated with a restriction enzyme as a vector. Thereafter, the target plasmid was obtained by the alkali method.

2-4 Construction of pQtTAW pETtTAW and pQCXIN (Clontech) constructed in 2-3 were each treated with restriction enzyme KpnI, fractionated and excised by 1% agarose electrophoresis, and Mag Extractor-PCR & Gel Clean up- Purification was performed. Ligation and transformation were performed using the pET tTA W fragment as an insert and the restriction enzyme-treated pET Blue2 as a vector. Thereafter, the target plasmid was obtained by the alkali method.

2-5 Construction of pQeGTREtTAW The pMSCVeGOVATREGITTAW constructed in 2-2 and the pQtTAW constructed in 2-4 were each treated with EcoRI, and fractionated and excised by 1% agarose electrophoresis, Mag Extractor-PCR & Gel Purification by Clean up- was performed. Ligation and transformation were performed using a fragment of about 1.5 kb obtained from pMSCVeGOVATREGITTAW as an insert and pQtTAW as a vector. Thereafter, the target plasmid was obtained by the alkali method.

2-6 Construction of pQeGOVATRETTAW The pQeGTREtTAW constructed in 2-5 and the pETOVA constructed in Example 1 were each treated with XhoI, and fractionated and excised by 1% agarose electrophoresis, Mag Extractor-PCR & Gel Purification by Clean up- was performed. Ligation and transformation were performed using the approximately 2.3 kb fragment obtained from pET OVA as an insert and pQeGTREtTAW treated with a restriction enzyme as a vector. Thereafter, the target plasmid was obtained by the alkali method.

2-7 Construction of pQeGOVA (DHSIII) TREtTAW Treat pQeGOVATERtTAW constructed in 2-6 with BglII, fractionate and excise the target fragment by 1% agarose electrophoresis, and purify by Mag Extractor-PCR & Gel Clean up- went. Self-ligation and transformation were performed using a fragment of about 8.7 kb obtained from pQeGOVATREtTAW as a vector. Thereafter, the target plasmid was obtained by the alkali method.

FIG. 2 shows a schematic diagram of the retroviral vector prepared in Example 2. FIG. 2 shows that the tTA fusion protein (tetR + VP16) is expressed, binds to the TRE sequence (tetra cyclin response element), and is transcribed from the downstream CMV minimal promoter (CMV mini = CMV minimum promoter).

(Example 3) Construction of viral vector for Ds-RED expression
3-1 Construction of pET DsRed DsRed was amplified by PCR from pIRES2-DsRed-Express (Clontech).
5 'primer (SEQ ID NO: 15)
3 ′ primer (SEQ ID NO: 16)
KOD-Plus-DNA Polymerase was used as the enzyme for the PCR reaction, and the conditions were as follows.
(1) 94 ° C. for 15 seconds, (2) 56 ° C. for 30 seconds, (3) 68 ° C. for 45 seconds, (1) to (3) for 30 cycles.
The solution after the PCR reaction was subjected to phenol chloroform extraction and ethanol precipitation.
Thereafter, DsRed and pET Blue2 amplified by PCR were treated with restriction enzymes with BamHI and MluI, respectively, fractionated and excised by 1% agarose electrophoresis, and purified by Mag Extractor-PCR & Gel Cleanup-. Ligation and transformation were performed using the DsRed fragment as an insert and the restriction enzyme-treated pET Blue2 as a vector. Thereafter, the target plasmid was obtained by the alkali method.

3-2 Construction of pQeGTREDsRedW The pETDsRed constructed in 3-1 and the pQeGTREtTAW constructed in 2-5 are treated with restriction enzymes with BamHI and MluI, respectively, and fractionated and excised from the target fragment by 1% agarose electrophoresis, Mag Extractor -Purification by PCR & Gel Clean up- was performed. Ligation and transformation were carried out using the DsRed fragment as an insert and pQeGTREtTAW treated with a restriction enzyme as a vector. Thereafter, the target plasmid was obtained by the alkali method.

Example 4 Production of Retrovirus First, GP-293 cells, which are virus-producing cells, were seeded in a 24-well collagen-coated tissue culture dish (IWAKI). 24 hours after seeding, when the cell density was 90 to 95%, cotransfection of the viral vector plasmid pQeGTREtTAW and pVSV-G constructed in 2-5 was performed using Lipofectamine 2000 (Invitrogen). Transfection was performed according to the protocol of Lipofectamine 2000, and DMEM containing 10 mM HEPES was used as the medium for transfection. Thereafter, the virus was concentrated by ultracentrifugation and used for injection experiments. In addition, the vectors of pQeGOVAtTAW constructed in 2-6 and pQeGTREDsRedW constructed in 3-2 were co-transfected with pVSV-G in the same manner as described above, and virus concentration was performed.

(Example 5) Production of transgenic chickens by microinjection and extraembryonic culture microinjection was carried out in accordance with the procedure described in International Publication No. 2004/016081 and Japanese Patent Application Laid-Open No. 2004-236626.
In order to analyze the expression of the tTA gene in a tissue-specific manner, the viral vector used for the injection was microinjected with the pQeGOVARETtTA retroviral vector obtained by the method described in Example 4 into the heart of a chicken fertilized egg for 55 hours, and then GO. Transgenic chickens were generated. As a control, a G0 transgenic chicken was prepared using pQeGTREtTAW retrovirus having no OVA sequence in the same manner.
In addition, two viral vectors, pQeGTREDsRedW and pQeGOVARETtTAW, which incorporate DsRed encoding a fluorescent protein as a target gene, were mixed and injected, and the tissue specificity of DsRed protein expression enhanced by tTA expressed specifically in the fallopian tube was analyzed. did.
In the post-injection extracorporeal culture method, G0 transgenic chickens were hatched in accordance with the procedures carried out in WO 2004/016081 pamphlet and JP 2004-236626 A.

(Example 6) Individual analysis
6-1 Induction with steroid hormones Transgenic chicken (G0 individual) obtained by microinjection and extraembryonic culture 2-week-old muscle, particularly pectoral muscle dissolved in 100% ethanol (DES: Sigma) By continuously administering 10 mg / ml at 0.1 ml / day for 10 days, the enormous part of the oviduct was enlarged by Estrogen induction. Thereafter, DES was injected again for one week or more after a state where no treatment was performed for 5 days or more, and dissection was performed.

6-2 Expression analysis Wild-type chickens and transgenic chickens that had been treated with hormones were dissected and each tissue was sampled. Total RNA and genomic DNA were obtained from the sampled tissue, and expression analysis of the target gene was performed by real-time PCR.

6-2-1 Acquisition of total RNA and cDNA synthesis by RT-PCR A very small amount of tissue was cut out from each tissue that had been thoroughly washed away with PBS, and was cut as finely as possible with scissors. Then, the total RNA concentration obtained using the QuickPrep Total RNA Extraction Kit (registered trademark, manufactured by TAKARA) was measured with a BioPhotometer (eppendorf), diluted to a constant concentration, and a reverse transcription reaction was performed. The procedure for obtaining RNA was in accordance with the protocol. Further, ReverseTra Ace (TOYOBO) was used for the reverse transcription reaction. A total of 12 μl of total RNA adjusted to a certain concentration and 1 μl of 10 pmol / μl Oligo (dT) were reacted at 70 ° C. for 10 minutes and allowed to stand on ice for 15 minutes. 4 μl of 5 × Buffer and 5 μl of 2 mM dNTPs were added to the solution, suspended well, and reacted at 42 ° C. for 5 minutes. Thereafter, 1 μl of ReverseTra Ace was added, a reverse transcription reaction was performed at 42 ° C. for 50 minutes, the enzyme was inactivated at 70 ° C. for 15 minutes, and the mixture was allowed to stand on ice for 10 minutes.

6-2-2 Acquisition of genome DNA First, a very small amount of tissue was cut out from each tissue that had been thoroughly washed with PBS, and was cut as finely as possible with scissors. Genome DNA was obtained using MagExactor-Genome-. The operation followed the protocol. After adding MagExactor-Genome-dissolution / adsorption solution to the tissue, the tissue mass is made very fine by using a 22G Terumo injection needle (TERUMO) and a Terumo syringe with a 26G injection needle for 1 ml tuberculin. Increased yield. Genome DNA concentration was measured with a BioPhotometer.

6-2-3 real-time PCR
Using the LightCycler FastStart DNA Master HybProbe (registered trademark, Roche), real-time PCR of the cDNA and the genomic DNA obtained in 6-2-1 and 6-2-2 was performed. The operation followed the protocol. The used chips, Eppendorf tubes, and MilliQ water for dilution were sterilized twice by autoclave and UV-sterilized overnight or longer. Pipetman used was dedicated to genome DNA not handling plasmids. Note that a set of tTA primer and probe was used for quantification of cDNA, and a set of packaging signal primer and probe capable of detecting a packaging signal was used for quantification of genome DNA.

-Real-time PCR conditions When the Mg2 + concentration was 4 mM and the annealing temperature was 58 ° C, specific and effective amplification was observed with the LightCycler. Real-time PCR was performed under these conditions. Both probes were used under the following conditions.
95 ° C., 10 min, 20 ° C./s 1 cycle
(Category 1) 95 ° C, 10s, 20 ° C / s (Category 2) 58 ° C, 15s, 20 ° C / s (Category 3) 72 ° C, 5s, 20 ° C / s 45cycle
40 ° C., 30 s, 20 ° C./s 1 cycle

・ Primer (packaging signal)
5 ′ primer (SEQ ID NO: 17)
3 ′ primer (SEQ ID NO: 18)

・ Hybridization probe (packaging signal)
3 ′ FITC (SEQ ID NO: 19) 5 ′ LCRed640 (SEQ ID NO: 20)

Primer (tTA) 5 'primer (SEQ ID NO: 21) 3' primer (SEQ ID NO: 22)

・ Hybridization probe (tTA) 3 'FITC (SEQ ID NO: 23) 5' LCRed640 (SEQ ID NO: 24)

<Samples> 2 μl of cDNA obtained by performing reverse transcription reaction with a unified RNA concentration of 800 ng was used as a sample, and 2 μl of genome DNA obtained from each tissue was diluted to 10 ng / μl and used as a sample. <Positive Control &Standard> Real-time PCR was performed by diluting the genomic DNA obtained from the clot of one copy of a fully transgenic chicken as a standard and a positive control in 6 steps. <Negative Control> Autoclave and UV-sterilized MilliQ water and wild type embryos, or genomic DNA obtained from wild type chicken clots were used. Mosaic rate calculation In order to unify the number of transgene copies in each tissue, real-time PCR was performed on the tissue genome and the mosaic rate was calculated to correct the mRNA expression level for each tissue.

6-2-5 Observation of tissue section with confocal microscope After fixing the oviduct part of the transgenic chicken in which hormone induction was performed in 6-1 with 3% formalin, the thickness was set to 20 μm with a razor, and the confocal microscope was used. (Olympus Fluoview FV1000) was observed. Individual samples # 219 and # 220 were used as samples.

Example 7 Further truncation of OVA promoter
7-1 Construction of pET- OVAΔPvuII The following vector was constructed to further cut the ovalbumin promoter sequence of about 2.3 kbp obtained in Example 1 and to further shorten it while showing tissue specificity. . PET-OVA obtained in Example 1-5 was digested with the restriction enzyme PvuII (TaKaRa), and the cleaved vector was self-ligated. This vector was digested again with the restriction enzyme PvuII, and then ligated using a 200 bp fragment obtained by digesting pET-OVA with the restriction enzyme PvuII as an insert. This vector was designated as pET-OVAΔPvuII.

7-2 Construction of pET-OVAΔBglIIXhoI After digesting the pET-OVA obtained in Example 1-5 with the restriction enzymes BglII (TaKaRa) and the restriction enzyme XhoI (TaKaRa), Klenow (TaKaRa) is obtained. It was used as a blunt end and then self-ligated. This vector was designated as pET-OVAΔBglIIXhoI.

7-3 Construction of pQeGOVA (ΔPvuII) TREtTAW and pQeGOVA (ΔBglIIXhoI) TREtTAW By the same method as in Example 2, pET- OVAΔPvuII obtained in 7-1 above and in 7-2 above The obtained pET-OVAΔBglIIXhoI was converted into a viral vector. The vectors were designated as pQeGOVA (ΔPvuII) TRETTAW and pQeGOVA (ΔBglIIXhoI) TRETTAW, respectively.

7-4 Production of retrovirus The pQeGOVA (ΔPvuII) TREtTAW and pQeGOVA (ΔBglIIXhoI) TRETTAW retroviral vectors were produced in the same manner as in Example 4.

7-5 Production of transgenic chicken A transgenic chicken was produced in the same manner as in Example 5 using the retroviral vector obtained in 7-4 above.

7-6 Individual analysis In the same manner as in Example 6, oviduct tissues were extracted from hormone-treated individuals, total RNA extraction and genomic DNA were extracted, and tetR / mosaic was calculated by real-time PCR (Fig. 7). ).

The schematic diagram of the ovalbumin promoter region used in the present invention is shown. FIG. 1a shows the ovalbumin promoter region (3.8 kbp) used in the examples of Japanese Patent Application Laid-Open No. 2004/236626. FIG. 1b shows the truncated ovalbumin promoter region (2.3 kbp) used in the present invention (Example 1). FIG. 2 shows a schematic diagram of the retroviral vector prepared in Example 2 incorporating the ovalbumin promoter region of FIG. 1b. The self-active vector structure is shown in which the ovibulin-specific expression by the ovalbumin promoter and the transcription enhancing effect by the transactivator are obtained. Tissues of individuals (# 2, # 205, # 21, # 5) produced by the retroviral vector of pQeGOVATRETTAW described in Example 5 and Example 6 and individuals (# 49) produced by the retroviral vector of pQeGTREtTAW Shows tetR / mosaic. The tTA mRNA was measured by real-time PCR (detection of the tetR sequence), and similarly the mosaic rate (detection of the packaging signal region) was calculated and corrected. In addition, it was confirmed in the same manner that the DES non-administered individual (# 2) was used as a control and it was not tubal-specific expression by DES. The vector structure for expressing DsRed as an exogenous target protein specifically in an oviduct is shown. The two viral vectors described were mixed and used for injection experiments to produce transgenic chickens. A tissue section of an individual hatched by double injection was confirmed with a confocal microscope (Example 6), and the fluorescence of the target DsRed was observed specifically in the fallopian tube tissue (individual # 219). As a control experiment, in the analysis of the individual injected with DsRed alone (individual # 220), the oviduct-specific tTA gene by the ovalbumin promoter did not function, and thus fluorescence could not be observed. From the above, it was demonstrated that the ovalbumin promoter used expresses the tTA gene specifically in the fallopian tube, and thereby expresses the target protein DsRed specifically in the fallopian tube. The tissue specificity of expression in individual # 219 was measured by real-time PCR. The schematic diagram and tetR / mosaic of the retroviral vector used for the production of the transgenic chicken in Example 7 are shown. OVA is an approximately 2.4 kbp ovalbumin promoter sequence obtained in Example 1 (SEQ ID NO: 2 in the Sequence Listing). OVA (ΔPvuII) is an ovalbumin promoter sequence of about 1.4 kbp obtained in Example 7-1 (SEQ ID NO: 3 in the Sequence Listing). OVA (ΔBglIIXhoI) is the approximately 1.0 kbp ovalbumin promoter sequence obtained in Example 7-2. Transgenic chickens prepared with viral vectors having each ovalbumin promoter sequence were treated with DES, and the oviduct tissue was excised, and tetR / mosaic was calculated. Expression in the oviduct cells was confirmed with the promoters of OVA and OVA (ΔPvuII).

Claims (13)

Transgenic birds produced by viral vectors, or
A method for producing a foreign target protein by a progeny transgenic bird obtained by mating the transgenic bird with another bird,
The viral vector is
A promoter for gene expression of the ovalbumin promoter,
a) it has a total parts of the hormone-dependent DNaseI hypersensitive site III,
b) does not contain hormone-dependent DNase I supersensitive site I and / or hormone-dependent DNase I supersensitive site II,
c) functioning specifically in the oviduct in birds, not exceeding 3 kbp and exceeding 1.0 kbp,
d) including the TATA box,
A promoter consisting of a partial region ,
Foreign target protein gene, and
Have a cis or transactivator system ,
The promoter induces oviduct-specific expression of the activator protein, and induces expression of a foreign target protein gene by the activator protein;
Production method of foreign target protein. The method for producing a foreign target protein according to claim 1, wherein the ovalbumin promoter is an avian ovalbumin promoter. The method for producing a foreign target protein according to claim 2, wherein the avian ovalbumin promoter is a chicken ovalbumin promoter. The method for producing a foreign target protein according to claim 3, wherein the chicken ovalbumin promoter is the chicken ovalbumin promoter described in SEQ ID NO: 1 in Sequence Listing. An activity in which the ovalbumin promoter consists of the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 2 and functions specifically in the oviduct The method for producing a foreign target protein according to any one of claims 1 to 3. An activity in which the ovalbumin promoter consists of the base sequence shown in SEQ ID NO: 3 or the base sequence shown in SEQ ID NO: 3 has 90% or more identity and functions specifically in the oviduct The method for producing a foreign target protein according to any one of claims 1 to 3. The method for producing a foreign target protein according to any one of claims 1 to 6, wherein the viral vector is a retroviral vector. The method for producing a foreign target protein according to any one of claims 1 to 7, wherein the viral vector further comprises a foreign target protein gene. The method for producing a foreign target protein according to any one of claims 1 to 8, wherein the viral vector further comprises an enhancer, a signal sequence, and a post-transcriptional regulator. The method for producing a foreign target protein according to any one of claims 1 to 9, wherein the transgenic bird is obtained by microinjecting a virus vector into a heart of an embryo at the stage of development of an avian fertilized egg and allowing it to hatch. The method for producing a foreign target protein according to any one of claims 1 to 10, wherein the progeny transgenic birds preferentially express the foreign target protein in a fallopian tube cell of a female individual. The method for producing a foreign target protein according to any one of claims 1 to 11, wherein the foreign target protein is an antibody, cytokine, insulin, hormone, bioactive protein, or a fusion protein comprising these. The method for producing a foreign target protein according to any one of claims 1 to 12, wherein the transgenic bird is a transgenic chicken.