WO2013031734A1 - 小胞体シャペロンプロモータを用いた外来遺伝子を発現するトランスジェニック鳥類 - Google Patents
小胞体シャペロンプロモータを用いた外来遺伝子を発現するトランスジェニック鳥類 Download PDFInfo
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- WO2013031734A1 WO2013031734A1 PCT/JP2012/071609 JP2012071609W WO2013031734A1 WO 2013031734 A1 WO2013031734 A1 WO 2013031734A1 JP 2012071609 W JP2012071609 W JP 2012071609W WO 2013031734 A1 WO2013031734 A1 WO 2013031734A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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- C12N2830/00—Vector systems having a special element relevant for transcription
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/90—Vector systems having a special element relevant for transcription from vertebrates avian
Definitions
- the present invention belongs to the technical fields of transgenic birds, methods for producing foreign useful proteins, and methods for producing transgenic birds.
- Patent Document 1 site-specific promoters of proteins localized in egg white such as ovalbumin promoter (Patent Document 1) and ovomucoid promoter (Patent Document 2) are often used.
- an actin promoter that is expressed throughout the body may be used (Patent Document 3).
- a promoter of a protein accumulated in egg white is a very effective promoter for inducing oviduct-specific gene expression and accumulating foreign useful proteins in egg white.
- the expression level of a foreign useful protein cannot be confirmed in blood other than the oviduct (Non-Patent Document 1, FIG. 3). This means that the production capacity of a chicken as an animal factory cannot be grasped until sexual maturity, and it is necessary to raise all the transgenic chickens until sexual maturity. Therefore, it takes a lot of time and labor to raise a low-productivity chicken until sexual maturity, which is a big problem.
- the purpose of the present invention is to produce a production amount of a foreign useful protein in egg white in transgenic birds with an expression level equal to or higher than when using an ovalbumin promoter or an actin promoter, and While achieving an expression level that can predict the expression level before sexual maturation, the expression level outside of the oviduct is suppressed to a low level, thereby reducing a large burden on birds.
- the inventors of the present invention are able to express a foreign useful protein in the egg white of an avian endoplasmic reticulum chaperone protein, and further to express the foreign useful protein in the egg white in an amount equal to or more than the ovalbumin or actin promoter.
- the expression level other than the oviduct can be suppressed to such a level that the expression level in the egg white can be predicted before the birds are sexually matured, and the burden on the birds can be reduced. It came.
- the present invention includes a nucleobase sequence in which (a) an avian endoplasmic reticulum chaperone promoter and (b) a nucleobase sequence encoding a foreign useful protein are functionally linked on the chromosome of a cell constituting the oviduct. It relates to transgenic birds.
- the endoplasmic reticulum chaperone promoter is preferably derived from chicken, more preferably has an ER stress response element motif, more preferably a gluco-regulated protein 78 promoter, or a protein disulfide isomerase promoter.
- the host is preferably poultry and more preferably a chicken.
- the foreign useful protein is preferably a cat-derived protein.
- the present invention also relates to a method for producing a foreign useful protein, comprising the step of recovering the foreign useful protein from the transgenic bird.
- the present invention also introduces a nucleobase sequence in which (a) an avian endoplasmic reticulum chaperone promoter and (b) a nucleobase sequence encoding a foreign useful protein are functionally linked onto the chromosome of a cell constituting the oviduct. And a method for producing a transgenic bird.
- the production method preferably further includes a step of selecting transgenic birds using the expression level of a foreign useful protein in blood as an index before sexual maturation.
- the host is preferably poultry and more preferably a chicken.
- the transgenic bird of the present invention can predict the expression level before spawning, the systemic expression level can be kept low, and ovalbumin
- the amount of foreign useful protein accumulated in egg white can be achieved to the same extent as in the case of using a promoter or actin promoter.
- the transgenic bird of the present invention comprises a nucleobase sequence in which (a) an avian endoplasmic reticulum chaperone promoter and (b) a nucleobase sequence encoding a useful foreign protein are functionally linked on the chromosome of a cell constituting the oviduct. including.
- Birds that serve as hosts for transgenic birds are not limited, but are preferably poultry birds that can be used as livestock.
- poultry birds include chickens, quails, turkeys, ducks, ostriches and ducks.
- Chickens and quails are particularly preferred because they are readily available, are plentiful as egg-laying species, have large eggs, and have established a method for safe large-scale breeding. Of these, chicken is most preferable.
- transgenic birds include G0 transgenic chimeric birds, G1 transgenic birds, and G2 and subsequent generations of transgenic birds.
- the endoplasmic reticulum chaperone promoter is not particularly limited as long as it is a promoter sequence having a function of expressing an endoplasmic reticulum chaperone protein, preferably an avian endoplasmic reticulum chaperone promoter, more preferably a host-derived endoplasmic reticulum chaperone promoter, Most preferred is an endoplasmic reticulum chaperone promoter derived from chicken.
- the endoplasmic reticulum is one of the organelles found in eukaryotic cells, and most of secreted and membrane proteins are synthesized here. It is also important as a field for folding the synthesized protein. There are chaperones involved in the folding of these secreted proteins and membrane proteins in the endoplasmic reticulum. Examples of endoplasmic reticulum chaperone proteins include Glucose-Regulated Protein Protein 78 (GRP78 / BiP), protein disulfide isomerase (PDI), and the like.
- GRP78 / BiP Glucose-Regulated Protein Protein 78
- PDI protein disulfide isomerase
- GRP78 plays a central role in protein transport in the endoplasmic reticulum and in the endoplasmic reticulum quality control mechanism. As part of its function, it also functions as a chaperone that binds to nascent proteins and suppresses aggregation to promote folding.
- PDI is an oxidoreductase present in the endoplasmic reticulum. Protein folding is catalyzed by the formation and exchange of protein disulfide bonds. Moreover, it has a role as a chaperone for forming a higher-order structure of a protein not having a disulfide bond as well as a denatured protein having a non-natural type disulfide bond.
- PDI is highly expressed in organs such as liver, thyroid gland, stomach, etc., but is hardly expressed in muscle and heart (J. Histochemistry and Cytochemistry 1996 44, 751). However, there was no suggestion or description that the endoplasmic reticulum chaperone protein is expressed in avian oviduct cells that produce egg white protein. As described above, the endoplasmic reticulum chaperone plays an important role in the cell.
- the promoters of GRP78 and PDI endoplasmic reticulum chaperones have a motif called ERSE (ER stress response element).
- ERSE ER stress response element
- the term “promoter” as used herein refers to a region on DNA that determines the start site of gene transcription and directly regulates its frequency. Promoters also include enhancers that promote transcription from the promoter.
- the ERSE motif includes nine arbitrary base sequences between the CCAAT and CCACG sequences and is represented as CCAATN 9 CCACG. Since the transcriptional activity drops when this sequence differs by only one base, it is considered to be an important motif for these promoters (J. Biological Chemistry 1998 273 33741).
- Examples of the endoplasmic reticulum chaperone promoter known to have the ERSE motif in addition to the two endoplasmic reticulum chaperone promoters include calreticulin. It is well known to those skilled in the art that any endoplasmic reticulum chaperone promoter having such an ERSE motif can have the same functions as GRP78 and PDI.
- a primer consisting of the base sequence of SEQ ID NO: 3 in the sequence listing and a primer consisting of the base sequence of SEQ ID NO: 4 in the sequence listing are used and amplified by PCR using the white legphone genome as a template. Sequence included in the base sequence. Specific examples include the base sequence of SEQ ID NO: 1 in the sequence listing.
- a primer consisting of the base sequence of SEQ ID NO: 5 in the sequence listing and a primer consisting of the base sequence of SEQ ID NO: 6 in the sequence listing are used and amplified by PCR using the white legphone genome as a template.
- Sequence included in the base sequence Specifically, for example, the base sequence of SEQ ID NO: 2 in the sequence listing can be mentioned.
- the avian endoplasmic reticulum chaperone promoter may be a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA described in SEQ ID NO: 1 or 2 in the sequence listing.
- the avian endoplasmic reticulum chaperone promoter may be DNA having 85% or more sequence identity to the base sequence described in SEQ ID NO: 1 or 2 in the sequence listing.
- DNA that hybridizes under stringent conditions with a DNA complementary to the DNA shown in SEQ ID NO: 1 or 2 in the sequence listing means the nucleotide sequence shown in SEQ ID NO: 1 or 2 in the sequence listing. It means DNA obtained by using colony hybridization method, plaque hybridization method, Southern hybridization method or the like under stringent conditions using DNA consisting of a complementary base sequence as a probe.
- DNA that hybridizes under stringent conditions means, for example, a high-temperature DNA at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. After hybridization, the filter is washed at 65 ° C. using a 2 ⁇ concentration SSC solution (the composition of the 1 ⁇ concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). The DNA which can be mentioned is mentioned.
- hybridization conditions have been described as described above, the conditions are not particularly limited.
- a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.
- the DNA that can hybridize under the above conditions is 70% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95%, with the DNA shown in SEQ ID NO: 1 or 2. % Or more, most preferably 98% or more of DNA.
- sequence identity means that the two DNAs 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, 575 Science Drive Medison, Wisconsin 11, USA 5; 37 Rice, P.M. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Center, Hinxton Hall, Cambridge, CB10 1RQ, England), and the ExPASy World Wide Web server for molecular biology (Geneva University Hospital and University of Geneva, Geneva, Switzerland).
- the foreign useful protein is not particularly limited, and secretory proteins, antibodies useful in the protein industry, bioactive proteins, enzymes, and the like can be used. Examples thereof include secretory proteins such as erythropoietin, G-CSF, thrombopoietin, and interferon, human monoclonal antibodies, chimeric antibodies, and artificial antibodies that have been modified such as single chain. Protein origins include livestock, poultry, pets, and laboratory animals. Examples of livestock include cattle, horses, pigs, goats, sheep, wild boars, inobuta, reindeer, deer, camels, donkeys, llamas, rabbits, minks, alpaca, buffalos and yaks.
- secretory proteins such as erythropoietin, G-CSF, thrombopoietin, and interferon
- human monoclonal antibodies chimeric antibodies
- artificial antibodies that have been modified such as single chain.
- Protein origins include livestock, poultry, pets, and laboratory
- Poultry includes chickens, ducks, geese, ostriches, quails, turkeys, pigeons and pheasants.
- pets include dogs, cats, rabbits, guinea pigs, rats, mice, hamsters, ferrets, parakeets, parrots, foxes, raccoons, monkeys, nine-birds, juvenile pine, wild birds, and pigeons.
- Examples of experimental animals include rats, mice, rabbits, dogs, cats, pigs, guinea pigs, monkeys, marmosets, gerbils, ferrets, cows, goats, sheep, chickens, and quails.
- Specific examples of the foreign useful protein include cat-derived erythropoietin.
- a foreign useful protein is not only a protein other than the transgenic bird to be produced, but also a protein originally possessed by an individual used to produce a transgenic bird, because a new base sequence is introduced into the genome of the individual. Corresponds to useful proteins.
- a nucleobase sequence encoding the foreign useful protein is functionally linked downstream of the promoter.
- Functionally linked means that various regulatory elements of a promoter that regulates the expression of a gene and a nucleobase sequence that encodes a protein are linked in a state where they can operate in a host cell.
- the boundary of the coding sequence of the foreign useful protein to be expressed is determined by the start codon at the 5 'end and the end codon at the 3' end corresponding to the start codon. It is preferred that the 5 'terminal vicinity of the start codon contains a Kozak consensus sequence.
- the nucleobase sequence operably linked to the ER chaperone promoter of the birds and the foreign useful protein is: Included on the chromosomes of cells throughout the body.
- the transgenic bird when the transgenic bird is a G0 transgenic chimeric bird, the nucleobase sequence is contained on the chromosomes of some cells constituting the oviduct and on the chromosomes of some cells throughout the body. It is. Thereby, the foreign useful protein can be accumulated in the egg laid by the transgenic bird and expressed to the extent that it can be detected in the blood.
- the present invention also includes a step of introducing a nucleobase sequence operably linked to an avian endoplasmic reticulum chaperone promoter and a nucleobase sequence encoding a foreign useful protein onto the chromosome of a cell constituting the oviduct.
- the present invention relates to a method for producing transgenic birds.
- Retroviral vectors include different forms of plasmids, viral particles, and packaging cells.
- a packaging cell is a cell into which a gene encoding at least one protein necessary for viral particle replication has been introduced.
- the retroviral vector used in the production of transgenic birds lacks replication ability.
- any of proteins (group specific antigen, gag), reverse transcriptase (polymerase, pol) and envelope glycoprotein (envelope, env) included in the inner core necessary for virus particle replication It is preferable that at least a part or all of the coding sequence or a sequence necessary for expression is deleted or a substitution or insertion mutation is made so that they cannot be expressed so that any one or a combination thereof is not expressed. Since retroviral vectors are limited by the length of the gene that can be inserted for each virus, the mutation is preferably a deletion mutation.
- the retroviral vector preferably contains a viral packaging signal (phi) that functions as a marker packaged in the viral particle. Since a part of the gag region may function as a virus packaging signal, it is preferable that the virus vector contains at least a part of the gag region that cannot be expressed (J Virol. 1987 61 (5): 1639).
- phi viral packaging signal
- the virus derived from Moloney murine leukemia virus, Moloney murine sarcoma virus, avian leukemia virus (ALV), human immunodeficiency virus (HIV), etc. are mentioned.
- a virus that is highly infectious to embryo cells and stem cells such as mouse stem cell virus and mouse embryonic stem cell virus (MSCV). More preferred is MSCV.
- a replication-defective retrovirus vector preferably contains a sequence derived from these viruses.
- VSV-G envelope protein derived from bovine vesicular stomatitis virus as the coat protein, but it is not limited to this virus type.
- the replication-defective retroviral vector may contain a transcription enhancer and / or a regulatory element.
- a transcription enhancer is a sequence that promotes transcription from a promoter gene, and is a gene sequence that cannot cause transcription by itself.
- the enhancer is not particularly limited, and examples include simian virus 40 (SV40), cytomegalovirus (CMV), thymidine kinase, steroid response element and lysozyme enhancer.
- a regulatory element is a gene sequence that contributes to transcriptional regulation and stabilization of RNA after transcription and cannot cause transcription by itself.
- the regulatory element is not particularly limited, and examples thereof include a woodchuck hepatitis virus-derived regulatory element (woodchucks post-translational regulatory element: WPRE, US Pat. No. 6,136,597).
- the retroviral vector includes at least a part of a long repetitive sequence (LTR) at the 5 'end and 3' end. Since LTR has a transcription promoter gene and a polyA addition signal, it can be used as a promoter or terminator gene.
- LTR long repetitive sequence
- a gene encoding a foreign useful protein, a promoter, a transcription enhancer, and / or a regulatory element is included between the 5 ′ LTR and the 3 ′ LTR, and the gene of the foreign useful protein is functionally linked downstream of the promoter.
- the nucleic acid sequence is Functionally linked means that the gene is operably linked to various regulatory elements of the promoter that regulates the expression of the gene and the gene. It is well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.
- no terminator or polyA addition signal be included between the 5 'LTR and the 3' LTR.
- the retroviral vector may contain a marker gene.
- a marker gene is a gene that encodes a protein that serves as a marker for identifying and isolating correctly transfected cells. No particular limitation is imposed on the marker gene, green fluorescent protein (GFP), a gene encoding a fluorescent protein such as cyan fluorescent protein (CFP), luciferase, neomycin resistance (neo r), hygromycin resistance (Hyg r), drug resistance gene such as puromycin resistance (Puro r), thymidine kinase, and a gene encoding such ⁇ - galactosidase.
- the marker gene preferably has a general promoter and elements necessary for expression.
- a replication-defective retrovirus vector lacks the gag, pol and env genes required for replication.
- a replication-defective retrovirus vector can be prepared by the following method. That is, a replication-deficient retrovirus plasmid capable of expressing a target protein and a VSV-G expression plasmid are simultaneously introduced into packaging cells carrying the gag and pol genes, and the culture supernatant is used as a virus solution. Alternatively, desirably, the VSV-G expression plasmid is introduced into the packaging cells infected with the virus solution, and the culture supernatant can be obtained as the virus solution. However, the method for obtaining the virus solution is limited to these methods. It is not a thing.
- the titer (titer) of the replication-defective retrovirus vector in the virus solution is not particularly limited as long as it retains the infectivity, but preferably 1 ⁇ 10 8 to 1 ⁇ 10 14 cfu / ml after centrifugal concentration. 1 ⁇ 10 9 to 1 ⁇ 10 14 cfu / ml is more preferable.
- the titer of the virus solution is defined by the number of cells infected when the virus solution is added to NIH3T3 cells (American Type Culture Collection CRL-1658). Specifically, 1 ⁇ l of virus solution was diluted in 2 ml medium, and this diluted solution was diluted 10 1 to 10 5 times. These dilutions are added to 1.5 ⁇ 10 4 NIH3T3 cells present in each well of a 6-well culture plate, and the percentage of cells expressing the marker gene neomycin resistance gene is examined from the resistance to G418. To measure the titer of the virus solution.
- Transgenic birds are preferably obtained by a method comprising infecting a bird embryo with a replication-defective retrovirus vector containing a promoter and a foreign gene, and hatching the embryo.
- Embryos are early in the ontogeny of multicellular organisms, and are encased in eggshells or egg membranes, but are in the mother's body and before eating food independently. Hatching is coming out of the eggshell or egg membrane and becoming independent of food.
- the avian embryo that infects the replication-defective retrovirus vector is not particularly limited, but it is preferable that 24 hours or more have passed since the start of incubation. More preferably, the embryo is 32 hours to 72 hours before the start of incubation. More preferably, the embryo is 48 hours to 64 hours before.
- the place where the virus is infected, that is, the place where the virus solution is introduced is not particularly limited, but is preferably in the heart or blood vessel formed in the embryo. In producing a G0 transgenic chimeric bird having high gene transfer efficiency, it is preferable to introduce a gene at an early stage where the heart beat can be observed. Examples of such a stage include a stage of 6 hours or less after the heart starts to beat. This stage is preferred because it carries the gene in the whole body in the bloodstream and the number of cells is small.
- Incubation refers to keeping fertilized eggs immediately after laying and avian fertilized eggs stored in an environment that cannot be generated immediately after laying in a developable environment.
- the optimum temperature for incubation is 37.2 to 37.8 ° C in a three-dimensional incubator (38.9 to 39.4 ° C at the top of the egg in a flat incubator), and the optimum humidity is 40 to 70 % Environment, but not limited to this environment.
- eggs are turned during incubation.
- the turning angle is preferably 30 ° or more and twice or more a day, but is not limited thereto.
- Infection of an avian embryo with a virus is preferably performed by microinjection in order to introduce a virus solution into a specific place such as the heart or blood vessels.
- Microinjection is a method in which a virus solution is directly introduced into a specific location using a micro glass tube having a thin tip under a microscope.
- the transgenic birds are preferably selected using the expression level of foreign useful protein in blood as an index before sexual maturation.
- a G0 transgenic chimeric bird By infecting a bird embryo with a replication-defective retrovirus vector containing a foreign gene that can be expressed by the endoplasmic reticulum chaperone promoter, and hatching the embryo, a G0 transgenic chimeric bird can be obtained.
- a G1 transgenic bird can be obtained by crossing a G0 transgenic chimeric bird having a foreign gene in a germ cell with a wild type bird or a G0 transgenic chimeric bird and selecting hatched chicks.
- G0 transgenic birds the probability that a foreign gene has been introduced into all cells is low, and in most cases, a cell that has been introduced with a foreign gene is different from a wild type cell that has not been introduced with a foreign gene. It is a G0 transgenic chimeric bird in which types of cells coexist in the same individual. When the transgenic bird is a G0 transgenic chimeric bird, a foreign gene has been introduced into the cells constituting the oviduct. On the other hand, G1 transgenic birds have foreign genes in all cells.
- introduction of a foreign gene into somatic cells or germ cells can be confirmed by collecting DNA or RNA from blood, somatic cells, sperm, etc. and performing operations such as PCR.
- the expression level of the target protein can be determined by subjecting plasma and organ debris to ELISA, electrophoresis, Western blot, measurement of target protein activity, and the like.
- the G2 and subsequent generations of transgenic birds can be produced by crossing G1 transgenic birds with wild-type birds, G0 transgenic chimeric birds, or G1 transgenic birds.
- G1 transgenic birds In the mating type, it is preferable from the viewpoint of efficiency that the female is mated with the G1 transgenic male.
- the present invention also relates to a method for producing a foreign useful protein, comprising a step of recovering the foreign useful protein from a transgenic bird.
- the foreign useful protein can be obtained by recovering from the transgenic bird. More specifically, the target protein is extracted, purified, and activated from the blood, somatic cell, or egg of the produced transgenic bird. It collects by performing any of these, or its combination.
- the method used for extraction or purification is not particularly limited, and may be any of methods such as fractional precipitation, centrifugation, two-phase separation, ultrafiltration, membrane separation, chromatography, immunochemical method, crystallization, and / or the like. Or the method containing the combination is mentioned.
- the ratio of the foreign protein expression level in blood / egg white expression level is preferably 1/1000 or more, more preferably 1/500 or more, and 1/120 or more. Is most preferred. Moreover, it is preferably 1/6 or less, more preferably 1/15 or less, because an individual expressing a foreign protein can be identified without imposing a burden on the host. / 31 or less is most preferable.
- the amount of foreign protein expressed in blood is preferably 700 pg / ml or more, and most preferably 0.3 ⁇ g / ml or more because foreign protein can be detected in blood. Moreover, it is preferably 10 ⁇ g / ml or less, more preferably 7 ⁇ g / ml or less, and most preferably 3.7 ⁇ g / ml or less because it reduces the burden on the host.
- the condition of the expression level of the foreign protein in egg white is not particularly limited, but is usually 1 mg / ml or less.
- the present inventors succeeded in producing a transgenic chicken expressing a cat-derived erythropoietin by an endoplasmic reticulum chaperone protein promoter.
- a transgenic chicken expressed in the actin promoter having a cat-derived erythropoietin concentration of 1.5 ⁇ g / ml from blood collected within 1 month after hatching Japanese Patent Application Laid-Open No. 2007-89578
- the expression level in the blood was reduced to 1/10 or less.
- the production amount in egg white is 193 ⁇ g / ml, which is reported in the ovalbumin promoter (Proc. Natl. Acad. Sci.
- the expression level in egg white can be predicted before sexual maturation by measuring the expression level in blood.
- Sexual maturation indicates that an animal is in a reproductive state. In the case of chickens, sexual maturity is reached and egg laying begins about 6 months after hatching.
- Example 1 (Step 1) Preparation of feline erythropoietin gene expression vector construct expressed by GRP78 promoter Blood was collected from the wing vein of white leghorn, and a genome was prepared using MagExtractor Genome (manufactured by Toyobo Co., Ltd.).
- the amplified fragment of about 3.4 kb was cleaved with EcoRI (manufactured by Takara Bio Inc.) and BamHI (manufactured by Takara Bio Inc.).
- the vector construct for inserting the promoter was removed by cleaving the actin promoter portion of pMSCVneobactfEPOwpre (Japanese Patent Laid-Open No. 2007-89578) previously prepared by digesting with EcoRI and BamHI, and this site was amplified by PCR and the fragment digested with the restriction enzyme was connected. Then, pMSCVneoGRPfEPOwpre was prepared.
- the DNA sequence cloned by PCR was also determined. The determined DNA sequence is shown in SEQ ID NO: 1 in the sequence listing.
- Step 2 Preparation of a feline erythropoietin gene expression vector construct expressed by the PDI promoter
- a synthetic primer for amplification of the PDI promoter (PDI3-5KD shown in SEQ ID NO: 5 in the sequence listing: 5'- Using AATGAATTCCCAGGCCAGACCCCCATAAC-3 ′ and PDI3-5KR: 5′-AAATACGATGAGAGCTGCGCTCCCTCTTCG-3 ′ shown in SEQ ID NO: 6 in the sequence listing, using the white legphone genome prepared by the above method as a template, KOD FX (Toyobo Co., Ltd.) PCR) was performed using The amplified fragment of about 3.5 kb was cleaved with EcoRI (manufactured by Takara Bio Inc.) and ClaI (manufactured by Takara Bio Inc.), inserted into the EcoRI, ClaI site of pBluescriptKS- (
- the prepared pBluPDI was cleaved with EcoRI and SalI (manufactured by Takara Bio Inc.), and a fragment of about 3.5 kb was purified.
- PMSCVneobactfEPOwpre was used as the vector construct for inserting the PDI promoter.
- the actin promoter part of this vector construct was removed by digestion with EcoRI and XhoI, and a 3.5 kb fragment digested with EcoRI and SalI was inserted into this site to prepare pMSCVneoPIfEPOwpre.
- the DNA sequence cloned by PCR was also determined. The determined DNA sequence is shown in SEQ ID NO: 2 in the sequence listing.
- Step 3 Preparation of retroviral vectors using pMSCVneoGRPfEPOwpre and pMSCVneoPIfEPOwpre and pVSV-G Unless otherwise specified, the medium contains 10% fetal bovine serum (Fetal Bovine Serum, FBS) and 50 unit / ml penicillin streptomycin. Method Eagle medium (Dulbecco's Modified Eagle Medium, DMEM) was used (Gibco). The culture was performed at 37 ° C. and 5% CO 2 . Endo Free Plasmid Maxi Kit (manufactured by Qiagen) was used for purification of the vector construct used for the retroviral vector.
- Method Eagle medium Dulbecco's Modified Eagle Medium, DMEM
- the retrovirus vector of pMSCVneoGRPfEPOwpre was prepared by the following procedure.
- packaging cells GP293 having gag and pol genes were placed in a collagen-coated culture dish having a diameter of 100 mm and 5 ⁇ 10 6 cells / dish. The seeding was carried out so as to be (70% confluent). The next day, the medium was removed and replaced with 7.2 ml medium. 56 ⁇ l of Lipofectamine. 2000 (manufactured by Invitrogen) was suspended in 1.4 ml of Opti-MEMI medium (manufactured by Gibco) and placed at room temperature for 5 minutes.
- the culture supernatant was passed through a 0.45 ⁇ m cellulose acetate filter (manufactured by Advantech) and collected in a centrifuge tube. Using an ultracentrifuge CS100GXL (manufactured by Hitachi Koki Co., Ltd.), the mixture was centrifuged at 28,000 rpm (5,000 g) for 1.5 hours. Remove the supernatant, add 20 ⁇ l of TNE buffer (50 mM Tris-HCl (pH 7.8), 130 mM NaCl, 1 mM EDTA) to the precipitate, suspend well, and centrifuge at 12,000 rpm for 1 minute in a small high-speed centrifuge.
- TNE buffer 50 mM Tris-HCl (pH 7.8), 130 mM NaCl, 1 mM EDTA
- the supernatant was passed through a 0.45 ⁇ m Durapore Ultra Free Filter (manufactured by Advantech) to obtain a virus solution.
- the retrovirus vector of pMSCVneoPIfEPOwpre was prepared according to the above method.
- Step 4 Measurement of virus titer
- NIH3T3 cells were seeded in a 6-well culture plate at 1.5 ⁇ 10 4 cells / well and cultured.
- 1 ⁇ l of the prepared virus solution was suspended in a medium containing 2 ml of polybrene (final concentration 8 ⁇ g / ml).
- the virus dilution was diluted 10 5 times 10 1 polybrene containing medium, a medium in culture the cells and replaced with virus dilution medium, were infected with virus NIH3T3 cells.
- the day after the infection the medium was replaced with a medium containing 1 mg / ml G418 (manufactured by Sigma).
- the medium was changed every other day with a medium containing G418 for 1 week.
- the plate was stained with 0.5% crystal violet (manufactured by Nacalai Tesque) ethanol solution, the number of colonies obtained was measured, and the titer was determined.
- Step 5 Selection of stable packaging cells
- 24-well culture plates were seeded with GP293 cells at 1 ⁇ 10 4 cells / well and cultured.
- the medium was replaced with 1 ml of a medium containing 8 ⁇ g / ml of polybrene. This was infected with the virus solution prepared in step 3.
- the cells were cloned by limiting dilution.
- the next day the cells were trypsinized, and the cell suspension was diluted with 1 mg / ml G418-containing medium to 13.3 cells / ml, and 150 ⁇ l was dispensed into a 96-well culture plate. did. This was cultured for about 1 to 2 weeks, 1 colony was grown in 1 well, and a fast-growing one was selected as a packaging cell clone.
- Step 6 Preparation of retroviral vector using pVSV-G in stable packaging cells
- Six to 7 ⁇ 10 6 stable packaging cells obtained in Step 5 were prepared in a collagen-coated culture dish having a diameter of 100 mm. Sowing. The next day, the medium was removed and 7.2 ml of medium was added. 56 ⁇ l of Lipofectamine. 2000 was suspended in 1.4 ml of Opti-MEMI medium and left at room temperature for 5 minutes. 24 ⁇ g of pVSV-G was suspended in 1.4 ml of Opti-MEMI medium. Lipofectamine. The 2000 solution and the DNA solution were mixed and placed at room temperature for 20 minutes. Thereafter, the whole amount was added to the culture dish and cultured for 6 hours.
- the medium was removed and 8 ml of medium was added. Thereafter, the cells were cultured for 48 hours.
- the culture supernatant was passed through a 0.45 ⁇ m cellulose acetate filter and collected in a centrifuge tube. Centrifugation was performed at 28,000 rpm (5,000 g) for 1.5 hours using an ultracentrifuge CS100GXL. Remove the supernatant, add 20 ⁇ l of TNE buffer to the precipitate, suspend well, centrifuge at 12,000 rpm in a small high-speed centrifuge for 1 minute, pass the supernatant through a 0.45 ⁇ m Durapore Ultra Free Filter and the virus solution. did. Thus, a virus solution having a titer of 1 ⁇ 10 8 or more was obtained.
- Step 7 Microinjection and artificial hatching of retroviral vectors into chicken embryos Microinjection and artificial hatching were performed under aseptic conditions.
- the outside of the chicken fertilized egg (Shiroyama breeding house) was sterilized with a disinfectant solution (manufactured by Showa Franchi) or 70% ethanol.
- Set the incubator P-008 (B) (manufactured by Showa Franchi Co., Ltd.) to an environment with a temperature of 38 ° C and a humidity of 50-60%. Incubation was performed while turning 90 ° each time.
- the egg was removed from the incubator and a hole of about 1 mm was made in the blunt end. Subsequently, a hole with a diameter of about 7 to 10 mm was made slightly above the center of the egg side.
- the virus solution prepared in step 6 was injected into the femtochip II (manufactured by Eppendorf), and about 2 ⁇ l of the virus solution was prepared by using femtojet (manufactured by Eppendorf). It was injected into the heart of a chicken embryo through a hole of about ⁇ 10 mm.
- the hole was closed with Scotch tape BK-15 (manufactured by Sumitomo 3M Co., Ltd.) and returned to the incubator to continue incubation.
- the incubator's turning was changed to 30 ° turning every 30 minutes.
- oxygen was supplied to the incubator at 60 cc / min for incubation.
- the eggs were turned and waited for natural hatching.
- Example 1 Production of transgenic chicken expressing feline EPO with actin promoter The same operations as in Steps 3 to 7 of Example 1 were carried out except that pMSCVneobactfEPOwpre (Japanese Patent Application Laid-Open No. 2007-89578) was used. A transgenic chicken expressing feline EPO with an actin promoter was produced.
- pMSCVneobactfEPOwpre Japanese Patent Application Laid-Open No. 2007-89578
- Example 1 Confirmation of expression in the blood and egg white of a feline-derived erythropoietin-expressing transgenic chicken
- the chick born in Step 7 of Example 1 was raised and grown.
- Infant SX Safety and Neo Safety 17 (Toyohashi Feed Co., Ltd.) were used as feed.
- Blood collection from the transgenic chicken was performed from the subwing vein.
- the collected blood was placed in an Eppendorf tube and allowed to stand at room temperature for 30 minutes or more, and then centrifuged at 4 ° C. and 3000 rpm for 5 minutes with a small high-speed centrifuge to separate serum and clot and use the supernatant as serum.
- the egg white was prepared by ultrasonication so that the whole was uniform.
- the activity of cat-derived erythropoietin was determined by a cell proliferation assay using Baf / EPOR, which is an EPO-dependent cell line (Japanese Patent Laid-Open No. 10-94393).
- the cell proliferation assay was performed using epodin (manufactured by Chugai Pharmaceutical Co., Ltd.) as a standard erythropoietin, and a calibration curve for the growth was drawn to determine the erythropoietin activity of an unknown sample.
- RPMI1640 liquid medium manufactured by Nissui Pharmaceutical Co., Ltd.
- FBS fetal bovine serum
- penicillin and streptomycin was used as a medium for Baf / EPOR cells.
- Epodin was added to a final concentration of 1 U / ml during normal Baf / EPOR cell culture. Cells in logarithmic growth phase were used for the cell proliferation assay.
- feline EPO concentration in blood and egg white of the transgenic chicken expressing feline EPO with the endoplasmic reticulum chaperone promoter of the example was measured. The results are shown in Table 1.
- the expression level of feline EPO in the blood by the endoplasmic reticulum chaperone promoter was about 1/10 or less of the blood expression level by the actin promoter.
- the expression level in the egg white of the transgenic chicken that expresses feline EPO with the endoplasmic reticulum chaperone promoter was equal to or higher than that of the transgenic chicken that expresses feline EPO with the actin promoter.
- Example 2 Measurement of Survival Rate until sexual Maturation of the Transgenic Chicken Prepared Feline EPO Expression Transformed by Endoplasmic Reticulum Chaperone Promoter Hatched by Steps 1 to 7 of Example 1 and Comparative Example 1
- a transgenic chicken and a feline-expressing transgenic chicken with an actin promoter were bred up to 6 months after hatching, and the number of transgenic chickens that died during this period was measured to determine the survival rate.
- Table 3 shows the results of the survival rate until sexual maturity.
- the survival rate until sexual maturity was 100% with the endoplasmic reticulum chaperone promoter, whereas it was 83% with the actin promoter, and when feline EPO was expressed at high levels throughout the body, the survival rate was high. It turns out that it falls.
- expression of foreign useful protein by ovalbumin or actin promoter can be expressed in the egg white in an amount equal to or more than that of transgenic birds, and before the birds are sexually matured. While achieving an expression level that can predict the expression level in egg white, the expression level other than the oviduct can be kept low, and a large burden on birds can be reduced. Thereby, compared with a prior art, a very useful transgenic bird can be provided industrially.
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Abstract
Description
GCG Wisconsin Package(Program Manual for The Wisconsin Package,Version8,1994年9月,Genetics Computer Group,575 Science Drive Medison,Wisconsin,USA 53711;
Rice,P.(1996)Program Manual for EGCG Package,Peter Rice,The Sanger Centre,Hinxton Hall,Cambridge,CB10 1RQ,England)、及び、
the ExPASy World Wide Web分子生物学用サーバー(Geneva University Hospital and University of Geneva,Geneva,Switzerland)。
(工程1)GRP78プロモータで発現するネコエリスロポエチン遺伝子発現ベクターコンストラクトの作製
ホワイトレグホンの翼下静脈より血液を採取し、MagExtractor Genome(東洋紡績(株)製)を用いてゲノムを調製した。GRP78プロモータ領域をPCRで増幅させるため、GRP78プロモータの増幅用合成プライマー(配列表の配列番号3に示すdnaK3KD:5’-ATTGAATTCACACGGAACCCGTAAACCCG-3’、及び配列表の配列番号4に示すdnaK3KR:5’-ATTGGATCCTCGCCGCTCTGCCAAAAGA-3’)を用い、調製したゲノムをテンプレートとしてPCR酵素であるKOD FX(東洋紡績(株)製)を用いてPCRを行った。増幅した約3.4kbのフラグメントをEcoRI(タカラバイオ(株)製)とBamHI(タカラバイオ(株)製)で切断した。プロモータを挿入するベクターコンストラクトは以前作製したpMSCVneobactfEPOwpre(特開2007-89578号公報)のアクチンプロモータ部をEcoRIとBamHIで切断することで取り除きこのサイトにPCRで増幅し、制限酵素で切断したフラグメントをつないで、pMSCVneoGRPfEPOwpreを作製した。PCRでクローニングしたDNA配列の決定も行った。決定したDNA配列を配列表の配列番号1に示す。
PDIプロモータ領域をPCRで増幅させるため、PDIプロモータの増幅用合成プライマー(配列表の配列番号5に示すPDI3-5KD:5’-AATGAATTCCCAGGCCAGACCCCATAAC-3’、及び配列表の配列番号6に示すPDI3-5KR:5’-AATATCGATGAGAGCTGCGCTCCCTCTTCG-3’)を用い、上記の方法で調製したホワイトレグホンのゲノムをテンプレートにしてKOD FX(東洋紡績(株)製)を用いてPCRを行った。増幅した約3.5kbのフラグメントをEcoRI(タカラバイオ(株)製)とClaI(タカラバイオ(株)製)で切断し、pBluescriptKS-(ストラタジーン社製)のEcoRI,ClaIサイトに挿入しpBluPDIを作製した。作製したpBluPDIをEcoRIとSalI(タカラバイオ(株)製)で切断し、約3.5kbのフラグメントを精製した。PDIプロモータを挿入するベクターコンストラクトはpMSCVneobactfEPOwpreを用いた。このベクターコンストラクトのアクチンプロモータ部をEcoRIとXhoIで切断することで取り除きこのサイトにEcoRIとSalIで切断した3.5kbのフラグメントを挿入して、pMSCVneoPDIfEPOwpreを作製した。PCRでクローニングしたDNA配列の決定も行った。決定したDNA配列を配列表の配列番号2に示す。
以後特に記述のない限り培地は10%ウシ胎児血清(Fetal Bovine Serum,FBS)と50unit/mlのペニシリンストレプトマイシンを含むダルベッコ変法イーグル培地(Dulbecco’s Modified Eagle Medium、DMEM)を用いた(ギブコ社製)。培養は37℃、CO2 5%で行った。レトロウイルスベクターに用いるベクターコンストラクトの精製はEndo Free Plasmid Maxi Kit(キアゲン社製)を用いた。
pMSCVneoPDIfEPOwpreのレトロウイルスベクターの調製は上記の方法に準じて行った。
ウイルス液の力価は、NIH3T3細胞(アメリカン・タイプ・カルチャー・コレクション CRL-1658)にウイルス液を添加したときに感染した細胞の数を測定することによって定義した。ウイルス液1μlを2ml培地に希釈しこの希釈液を101から105倍に希釈した。これらの希釈液を、6ウェル培養プレートの各ウェルに存在する1.5×104個のNIH3T3細胞に加え、マーカー遺伝子であるネオマイシン耐性遺伝子を発現している細胞の割合をG418に対する耐性から調べることによりウイルス液の力価を測定した。105希釈で1コロニー現れた場合、ウイルス力価は1×105cfu/2ml×2000=1×108cfu/mlとなる。
ウイルス感染の前日に24ウェル培養プレートにGP293細胞を1×104個/ウェルとなるよう播種し、培養した。ウイルス感染当日8μg/mlのポリブレンを含有する培地1mlと培地交換した。これに、工程3で調製したウイルス液を感染させた。以後、細胞を限界希釈法によりクローン化した。具体的には、次の日、細胞をトリプシン処理し、1mg/mlのG418入り培地で13.3個/mlとなるように細胞懸濁液を希釈し、96ウェル培養プレートに150μlずつ分注した。これを1~2週間程度培養し、1ウェルに1コロニー生育し、増殖の速いものをパッケージング細胞のクローンとして選択した。
直径100mmのコラーゲンコートされた培養ディッシュに工程5で得られた安定パッケージング細胞を6~7×106個となるように播種した。次の日、培地を除き7.2mlの培地を加えた。56μlのLipofectamine.2000を1.4mlのOpti-MEMI培地に懸濁し、室温で5分間おいた。24μgのpVSV-Gを1.4mlのOpti-MEMI培地に懸濁した。Lipofectamine.2000溶液とDNA溶液を混合し、室温で20分間おいた。この後、培養ディッシュに全量加え6時間培養した。6時間培養後培地を除き8mlの培地を加えた。その後48時間培養した。培養上清を0.45μmのセルロースアセテートフィルターに通し、遠心管に集めた。超遠心機CS100GXLを用い、28,000rpm(5,000g)で1.5時間遠心分離した。上清を取り除き、沈殿に20μlのTNE緩衝液を加え、よく懸濁して小型高速遠心分離機12,000rpmで1分間遠心分離し、上清を0.45μmデュラポアウルトラフリーフィルターに通しウイルス液とした。このようにして1×108以上の力価を持つウイルス液が得られた。
マイクロインジェクションと人工孵化は無菌条件下で行った。ニワトリ受精卵(城山種鶏場)の外側を消毒液((株)昭和フランキ製)または70%エタノールで除菌した。孵卵器P-008(B)型((株)昭和フランキ製)を温度38℃、湿度50~60%の環境になるようにセットし、電源を入れた時刻を孵卵開始時刻とし、以後15分毎に90°転卵しながら孵卵を行った。
pMSCVneobactfEPOwpre(特開2007-89578号公報)を使用した以外は、上記実施例1の工程3~7と同様の操作を行い、アクチンプロモータでネコEPOを発現するトランスジェニックニワトリを作製した。
上記実施例1の工程7により誕生したヒナを飼育して成長させた。飼料として幼雛SXセーフティー及びネオセーフティー17(豊橋飼料(株)製)を用いた。トランスジェニックニワトリからの採血は翼下静脈より行った。採血した血液はエッペンドルフチューブにいれ、室温で30分以上放置した後、小型高速遠心分離機で4℃、3000rpmで5分間遠心分離を行い、血清と血餅を分離し上清を血清とした。卵白は超音波により全体が均一となるように調製した。
上記実施例1の工程1から7、及び比較例1に記載の方法で孵化させた小胞体シャペロンプロモータによるネコEPO発現トランスジェニックニワトリ、およびアクチンプロモータによるネコEPO発現トランスジェニックニワトリを、孵化から6ヶ月まで飼育し、この期間中に死亡したトランスジェニックニワトリの数を測定し生存率を求めた。測定した性成熟までの生存率の結果を表3に示す。
Claims (12)
- 輸卵管を構成する細胞の染色体上に、
(a)鳥類の小胞体シャペロンプロモータ、及び
(b)外来有用タンパク質をコードする核酸塩基配列
が機能的に連結した核酸塩基配列を含むトランスジェニック鳥類。 - 小胞体シャペロンプロモータがニワトリ由来である請求項1に記載のトランスジェニック鳥類。
- 小胞体シャペロンプロモータが、ER stress response elementモチーフを有する、請求項1又は2に記載のトランスジェニック鳥類。
- ER stress response elementモチーフを有する小胞体シャペロンプロモータが、glucose-regulated protein78プロモータ、又はタンパク質ジスルフィドイソメラーゼプロモータである、請求項3に記載のトランスジェニック鳥類。
- 宿主が家禽である請求項1~4のいずれかに記載のトランスジェニック鳥類。
- 家禽がニワトリである請求項5に記載のトランスジェニック鳥類。
- 外来有用タンパク質がネコ由来タンパク質である請求項1~6のいずれかに記載のトランスジェニック鳥類。
- 請求項1~7のいずれかに記載のトランスジェニック鳥類から外来有用タンパク質を回収する工程を含む、外来有用タンパク質の製造方法。
- 輸卵管を構成する細胞の染色体上に、
(a)鳥類の小胞体シャペロンプロモータ、及び
(b)外来有用タンパク質をコードする核酸塩基配列
が機能的に連結した核酸塩基配列を導入する工程を含む、トランスジェニック鳥類の作製方法。 - 更に、性成熟前に、血中の外来有用タンパク質の発現量を指標として、トランスジェニック鳥類の選別を行う工程を含む、請求項9に記載のトランスジェニック鳥類の作製方法。
- 宿主が家禽である請求項9又は10に記載のトランスジェニック鳥類の作製方法。
- 家禽がニワトリである請求項11に記載のトランスジェニック鳥類の作製方法。
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