[go: up one dir, main page]

WO2007066775A1 - OISEAUX TRANSGÉNIQUES AYANT UN RÉSIDU Gla DANS UNE PROTÉINE DÉRIVÉE D’UN GÈNE ÉTRANGER - Google Patents

OISEAUX TRANSGÉNIQUES AYANT UN RÉSIDU Gla DANS UNE PROTÉINE DÉRIVÉE D’UN GÈNE ÉTRANGER Download PDF

Info

Publication number
WO2007066775A1
WO2007066775A1 PCT/JP2006/324593 JP2006324593W WO2007066775A1 WO 2007066775 A1 WO2007066775 A1 WO 2007066775A1 JP 2006324593 W JP2006324593 W JP 2006324593W WO 2007066775 A1 WO2007066775 A1 WO 2007066775A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
blood coagulation
transgenic
gene
vii
Prior art date
Application number
PCT/JP2006/324593
Other languages
English (en)
Japanese (ja)
Inventor
Tomoyuki Nakaishi
Takuya Shindo
Kazuyoshi Kaminaka
Original Assignee
Kaneka Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kaneka Corporation filed Critical Kaneka Corporation
Publication of WO2007066775A1 publication Critical patent/WO2007066775A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins

Definitions

  • the method of producing tanks by organisms is capable of producing tanks in a strike because the microorganisms are fast and the growth is simple.
  • microorganisms often do not correct the target tank quality. Therefore, it is difficult to adequately measure the quality of tanks that have the same properties as natural ones, and the road to production is far away.
  • the mainstream method has been to introduce a target protein gene into a mammalian cell and produce the target protein by causing the cell to propagate.
  • Pharmaceuticals such as offspring, thrombolytic agents, and antibody drugs are produced, sold, and used in genetically engineered mammals.
  • the method using dairy cells requires a dedicated tank land, which requires a high cost.
  • the most common method is to use a twist vector. So far, studies using the tow vector of bloodstream disease () have been reported (). The tank is used as a racket, and the robot is used as a Cytomega Wis (C) robot. We have succeeded in the production of trans-kinds by introducing the TW vector of Stage X later. It was reported that the analysis method using the standard was ⁇ (white is 4), and the calculation based on the lactate property was ⁇ 3 ⁇ . In addition, it is the GO transgenic chimera 2 at that time. As a result of adjusting the percentage of vesicles, 5 degrees of GO transchimeric chimeras had an introduced gene in sperm.
  • a template such as Xa Xa or Xa Xa (where a is the active type and a is the precursor is a precursor) is used to open the child between 52 e 53. And become an active a child. It becomes more active when the child binds to the a child. As a result, a child has X or X sexualization, and Xa Xa etc. sexualization occurs. That's why this is chained and amplified.
  • active Xa child makes the putombin an active tombine, which in turn degenerates the nogen. The tin is polymerized to form a net and is completed.
  • blood is a tank that is very easily decomposed, and
  • the challenge is to provide new laws such as tanks including ranks.
  • To solve the problem 009 includes tanks containing Ga, tanks containing empty cells, and empty tanks containing empty materials. I thought about using transic for the production of tanks. Physically, the activity was used only after receiving Ga as the target tank. Modification of tank quality Even with the same individual, it depends on the weave. Furthermore, in taxonomically different birds, the offspring of mammals receive G a in order to be active, and
  • the transic acid-producing protein can be obtained.
  • 001 that is, at least one kind of tank selected from the group consisting of an active tank by Ga, a tank, and a tank quality containing a tank position has an exogenous gene containing the sequence. It is a class of transformers.
  • the introductory gene is an exogenous gene containing an active protein sequence according to G a, or a exogenous gene containing a protein or its protein sequence. It is an exogenous gene that contains a series of tanks including the pump position.
  • the protein activated by G a is a blood 11 X, an X-child, a protein C, a protein S, a protein Z, or a protein exhibiting substantially the same physical properties as those. It is preferable that the protein and its protein, as well as the protein containing the above protein, be blood 11 X, X, X X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X, X. X. It is preferable that the protein has a difference in tissue plasmon activator or a tank that exhibits substantially the same physical properties as those.
  • Ming trans genus It is preferable to obtain the Ming trans genus, and to obtain avian fertilized eggs by a method including infecting with a twis vector containing an exogenous gene after the first 24 hours and splenicing the embryo.
  • X-child a type 11 X, X-child, putein C and its body, which contains 5 to 5 o neuranic acid per o, and 2 to 4 o per o.
  • a preferred type of protein is selected from the group consisting of neuranic acid-containing sex forms X, X-children, putein C and their bodies.
  • the above-mentioned protein is preferably a protein selected from the active form and its body.
  • Ming translucent, at least one kind of tank selected from the group consisting of an active tank by G, a tank, and a tank quality containing a tank position has an exogenous gene containing the train. It is a thing.
  • a tank is a combination of the above two anoic acids, including what is commonly called o. Also, the anoic acid may be modified. In order to separate the quality of the Is preferred. Moreover, it is not limited to the column of secretory Guna self.
  • Ga is a 4-box (Y-box), which is a group in which a radical is added to the V element of glutamic acid. Ga means that one of the tank quality conversion tanks becomes Ga (becomes Ga).
  • Examples include, but are not limited to, C, protein S, protein Z, Oscan, Tox Ga, and the like. More preferably, it is a shift of blood 11 X, X child, protein C, protein S, protein Z, or a tank substance having substantially the same physical properties as those.
  • a tank exhibiting substantially the same physical properties as those described above is an amino acid sequence in which the above-mentioned Ga has an active amino acid or a few amino acids that are missing, added, or substituted. , A non-sanitary one is shown, and if the sanitary is the same, it is qualitatively the same regardless of gender.
  • a tank containing a sample is a tank containing Ag and N. It is more preferable that chains containing Ag and N are exposed on the surface of the three structures of the tank for the purpose of disconnection by the template. Exposed on the surface of the structure depends on the target tank, the reaction in a reaction that can act on the target, and the fact that the target material is cleaved by the target. Be adjusted.
  • a template is a template with a center at the active position. It is defined as the active form by being defined as a precursor, an active form or a precursor of the active form.
  • Cell pump Te as 11 X, X, X X terminal, flop click Lee down, plus down, up Thein C, plus Roh plasminogen, tissue plasminogen Roh Gen'akuchibe but data and the like, Do limited thereto.
  • the introductory gene is not particularly limited, and may include not only non-avian genes but also those of birds. Even if the sequence used by an individual used for the production of transic species is introduced into the original gene by the body, it is called an exogenous gene.
  • the exogenous gene contains a tank-like de-sequence, which is determined by the three-end corresponding to the five-end don of the sequence. It is preferable to include a consensus array of 5 ends. It is preferred that there be some ranks in the stream of trains.
  • the native gene preferably comprises some or all of the appropriate gene for expression in birds.
  • a gene is a region that determines the position of a gene and directly regulates its degree.
  • Congenital genes may include areas other than the above.
  • Twist vectors include the morphology of plus, wisteria, and packaging cells.
  • the packaging is a cell into which a gene that has at least one of the proteins necessary for the production of a virus has been introduced.
  • the Twist vector used in the Is preferred.
  • a tank (o S ec caea) or a reverse transcription (Po e ase o) vector tank (e eo e) contained in the internal resin necessary for the production of the Wi It is preferred that the combination be non-expressing, lack some or all of the sequences required for expression, or be substituted, such that they are non-expressing.
  • the twice vector the number of genes that can be used for each whistle is limited, the difference is preferred, and the safety and the length of the insert are increased.
  • the widget vector preferably contains a widget gna (P) that acts as a landmark to be tagged to the widget. From a whisker perspective, it is preferable that the whistle vector contains a part of the unexpressed a region, because it may function as a whisking guna in the a region (o 987 a 6 (5) 639 46.
  • Us virus examples thereof include Us virus, hematological disease virus (), human epidemics virus (), Us cell virus (SC), Us cell virus (S), and the like.
  • Us blood disease virus or us virus is preferred, but for birds, infectious cells of cells and cells such as us cell virus (SC) and us cell virus (S) may be used. I like it. More preferred is a replicating mouse cell (SC).
  • SC us cell virus
  • S us cell virus
  • SC replicating mouse cell
  • the Twist vector of Ming is preferably a systematic vector or one containing a systematic vector.
  • Naputa genes are those that are constant / specially active in birds.
  • the use of different genes reduces the possibility that the expression of the target protein will adversely affect the life and survival of the species. Or there is an advantage that can be eliminated.
  • na pat gene Although there is no particular limitation on the na pat gene, all na pat genes can be mentioned. It is often the case that maturity is driven by active activities.
  • genes are birds, ovatransun, ovoid, ovotin, zotim, G2 gn, G3 gn, ovo, ovogptain, ovoptain, ovogn. , Statin, and Avidi can be mentioned, and it is preferable to include all of them. It is particularly preferable to use these types of genes because they can develop the desired protein quality in eggs.
  • An unidentified vector gene is an unorganized vector gene. There is no particular restriction on the type of gene, but one that is present in almost all cells of birds. In that case, since the target protein is also expressed, it is possible to detect the presence or absence of the expression at the chick stage.
  • actin platter Although not limited to these types of genes, actin platter, platter gene, neptepta gene, nwis 4 (S 4) platter gene, cytomegavirus (C) platter. Genes, Rous Wis (S) Peta Genes Whisper Genes.
  • actin protein gene includes the Watt actin gene, and it is preferable that the above actin gene includes the Watt actin gene.
  • the 0200 Twist vector may include transcriptional sensors or adjustments.
  • a sensor is a region that promotes transcription of proteins, but is a region that does or does not cause transcription by itself. Connected to a different model than the one that is now functional In many cases, it works well, so there is no particular limitation on the combination with the computer. Examples of the sensor include, but are not limited to, S4C-derived sensors and steadiness sensors. Mento is a region that causes transcription, contributes to transcription, or does not transcribe by itself. Although the material is not particularly limited, wood yak equipment (o odc c os a sc oaeao ee e WP 6 3 6597 detailed book) and the like are preferable.
  • the twist vector used for clarity may include a gene.
  • a gene is a gene that carries a protein that serves as a marker for the fixed and separated cells that have been correctly transferred.
  • the gene is not particularly limited, gene genes (eo) quinine (eo) quinine (eo) quinine (e.g. )
  • gene genes eo
  • quinine eo
  • quinine e.g.
  • drug genes such as pin (P o)
  • genes such as claw, dodactase, anotranslase, clam acetranslase, lacta, and galactose are listed.
  • the mosquito gene preferably accompanies the pattem gene and the elements necessary for its realization.
  • the 002 vector contains a part of the long repeat sequence (o e a e e a) at the 5th and 3rd ends. Since it has a transcription pattern and o gene, it can be used as a pattern gene and a gene pattern.
  • the target protein, protein, transcriptional sensor or regulatory element is included between 5 and 3.
  • the vector and o-gun are not included between 5 and 3.
  • the duplicated twistors used in the description lack the o and e genes required for replication.
  • o Insert Twice Plus S G Plus capable of expressing the target protein into the casing cell that has the gene, and use it as the upper virus.
  • S G Plus is introduced into the casing cells infected with the above-mentioned Wis solution to obtain the upper virus. It is preferable to shrink the whiskey solution if necessary.
  • Twist Vector is not limited to this method.
  • the value of the Wis solution is defined by the number of infected cells when the Wis solution is added to 33 (American type YA-Chon C 658). Physically, it is a 6-up unit (an area of about 9
  • the value of the Wis solution is measured by adjusting the concentration of cells in which the diluted Wis solution diluted with 2 times is expressed in the cells that express the ion gene that is G4 8 (neoin). .
  • the infection of embryos with the replication Twist vector will be explained.
  • Embryos infected with the Twist vector are preferably older than 24 hours. More preferred are embryos between 32 and 72 years old. More preferred are embryos between 48 and 64 years old.
  • To The preferred location for introducing the maltwisvector that infects the whistle vector is in the heart or in the duct. It is clear that, in order to generate GO transgenic chimeras with a high gene rate, it is possible to introduce a gene at an early stage (within 6 seconds after the start of the beat of the spleen) where the heart can be observed. I like it. This is because they carry a gene to the whole body and have few cells.
  • 002 is the method for micro glass with a thin tip under a microscope, in which the whiskey solution is directly introduced into a specific place.
  • the injection is preferred to other genes such as ponkpon and other genes, because it introduces a wisdom fluid into a cardiovascular region.
  • the case is not particularly limited, and for example, if it is a horse, the degree is 37 2 to 37.8.
  • humidity is 4 to 7 degrees, which is the optimal environment for occurrence, but is not limited to this environment. Also of eggs.
  • birds to be used in 002 are poultry that can be used as livestock.
  • poultry examples thereof include wort ,,, ostrich, quail, ahi and the like.
  • wat is particularly preferable because it is easy to obtain and proliferates as eggs, and eggs are large and mass production methods are established.
  • GO transgenic chimeras can be obtained by infecting a twiz vector containing an exogenous gene of a bird as in 028 and spleenizing the embryo.
  • G transgenics can be obtained by selecting GO transgenic chimeras having an exogenous gene in germ cells and crossing with wild transgenics or GO transgenic chimeras.
  • GO transgenic chimera It is a low probability that an exogenous gene is introduced into all cells, and it is a chimera state in which a gene that is introduced into most wild-type cells coexists with the gene.
  • almost all G G It has the first introduced gene in each cell.
  • the PC can be used to intro- Jerusalem the genes of vesicles and germ cells, blood, sperm, and native vesicles. It can also be judged from the expression of the target protein. It can be adjusted depending on the emission of tank, S and electricity, and the activity of the target tank.
  • G transzic wild female, G transzic wild male, and G transzic female can be considered, and further, mating by offspring and their parents is possible. Above all, it is preferable from the point of view of efficiency because it is possible to cross a large number of G in the wild with G.
  • the method used for purification and purification is not particularly limited, and includes, for example, fractionation, centrifugation, two-phase, ultrafiltration, chromatography, immunological method, conjugation method or a combination thereof. And so on.
  • tanks containing G a may cause oxidization due to the presence of 2 ions, and the identities of the cations due to the absence of 2 cations are recognized and purified using those with the target alkanol. I like that.
  • You may also activate the target protein if necessary. In particular, if the target protein is a child, it may be activated appropriately as needed.
  • the precursor and / or active form depends on the degree of divalent gold ion selected from the lithium ion, the ion ion, the ion ion, the ion ion, the ion ion, and the ion ion for strontium.
  • the tannins of the 003 Akira transzic are the selected tannins consisting of 11 X, X children, ptain C and their bodies containing o-5 to o o neuranic acid per o,
  • the above-mentioned tank quality is preferably one that contains 2 to 4 o of neuranic acid.
  • the above-mentioned protein is preferably a protein selected from the active form and its body.
  • Ga has a tank, a separator, and a separator.
  • Transgenics that produce a protein containing a position and a method for producing the same are provided. As a result, it becomes possible to have a transgenic species that expresses precursors and, in some cases, precursors and their sex-type proteins before blood 11 X, X-cells, protein C, etc. At the same time, it has also become possible to produce trans-types that express a stable and stable protein with a position and a configuration.
  • SC eobac is based on (Ge ee 994 A (2) 368 ⁇ Internet (cb o), and completes C g (Zinc Access X 24) e (235). Entered between ⁇ P (628) (Yobo). This was cleaved with d (Takara Io) and recovered with ae P os a ase P (Takara Io) and e eac o Cea (QG). This was collected by gas chromatography and the target piece by e Ge ac o (QG) (Kuta).
  • C OPO puts blood clot (2) in G ⁇ ⁇ ⁇ G in the column of 5 c acccacc G Gc c ac (7 X is Sa X o position, upper case is start don don) in PC position of C OPO.
  • the fragment amplified by PC using p was recovered by ePCPfcao (QG) and cleaved by d (Takaraio). This was electrically collected with gas and the target piece was collected with eGe ac o (Q G) (Insert).
  • the Kuta-carried insert piece was ligated with a o e ⁇ 2 ⁇ (Takara Io), and this was transformed into ⁇ co 5a a Co e e Ce s (Takara Io). It was applied to the ground containing the replaced colon 5 and nourished with C for 2 to 5 times.
  • the cultivated rice was cultivated in the area containing 5 1 to 5 and was cultivated 6 to 2 times in C, and then the plus was extracted.
  • the plus was subjected to restriction treatment with co (Takara Io) and rubbed. I chose the plus that has the structure of SC eobac.
  • the inside gene be a ac aase, whisking guna, tissue eoc es sa ce ee, organized tissue be a cooe, and long repeat sequences 5 3 It all comes from SC eobac.
  • the diluted cells Suspended in the soil containing G4 8 and diluted so that it would be in the same soil.
  • the diluted cells are not put in 96 U (make one in the U), the cell is early, GP293 cells are selected and
  • the human stable stabilizing cells obtained in Example 3 were set to be 5X (7 nt) in the long-digested diet. It will be 9 minutes. The next day, the land was removed, and the land of 7.2 and 25 of u were added and further nourished. Fifty-six of ec a e 2 were suspended in O 1 of 41, and 5 (o ca e 2) 2 S G were suspended in 41 of earth at room temperature (). of. . . ⁇ Mixture of the 2nd solution and the positive solution was performed at room temperature. This was added to the dish and fed for 6 times. The land was removed, and g 1 land and 2 P S ffe So o were added and fed for 24 hours. Above ⁇ Passed through 45 sessator and collected by centrifugation. Using a heart machine, it was centrifuged at 28 (5) for 5 minutes.
  • the SC eobac obtained in 1. was cleaved with Ca (io), treated with a e Pos a ase P, purified, and collected.
  • the products used in the implementation were used. This was electrically charged with a gas, and the target piece was collected (5 ct). Synthetic octup of 2 5 ca ca.
  • the target piece was collected (5 inserts).
  • the 5 inserts were ligated and transformed into • co 5a a.
  • Stable coding cells and twist vectors can be prepared in the same manner as described above. If you use the WP series, it is easy to get a cheap price. It is thought that this is because sexuality is strengthened by the WP series and is stabilized.
  • the value of the Wis solution was defined by the number of infected cells when the Wis solution was added to 33 (American type YA-Chon C 658). 2 out of 5 x 3 3 cells in 6 up (corresponding to an area of about 9.4c)
  • the titer of the Wis solution was determined by adjusting the ratio of the Wis solution diluted with 4 times the dilution of the Wis solution to the cells expressing the O.
  • the whisker number is cf if you give a 4 on a bake.
  • the time at which was entered was set as the start time (), and after that, the procedure was performed.
  • the eggs were taken out from the machine and cut with a ta (Pukson), which had a diameter (3, 5c) and a diamond (2, mat 2, 35) attached.
  • the wat () was cut to a diameter of 4.5 c, the fertilized egg whose contents were discarded was transferred, and the injected embryo was moved upward.
  • Microstem Stem SZX inject the whistle solution into Mutip 11 (Choppend), and use Mu (Choppend) to put Wis 2 of Run 4 (WP) or Run 5 (WP) into the heart. I got an inquiry.
  • This hole is closed with (Saran Lat Co., which is cut into c with white glue).
  • Example 7 The chicks born in Example 7 were bred and grown. As a fee, x Setty Setty (made by Tory Company) was used. Cunnadium hydrate () 3 8 was used as a solution, and it was used as a 3.8 kunnadium solution.
  • the child's deprivation law was used.
  • the sump was diluted with saline (P S).
  • Tobo S (Dadebing) and child deficiency (Joji King) were used as drugs, and C (Dadebing) was used as a measuring instrument, and the sample sex was calculated from that of normal humans. Human. When calculating the amount, it was calculated that the degree of humans was • 5x1.
  • Figure 3 shows the results of blood molecules in GO Transic. Injection (from the 7th of) is the GO Transic of the born chick.
  • the number of the first acquisition of 3 shown in 3 is shown.
  • 3 is the individual obtained in the third acquisition.
  • the sump is a P S containing electricity, P transferred to the P membrane under the conditions of e-di 25 (at), K, and 5 ee 2.
  • the width of a piece of b corresponding to the a region of PC and the twisvector can be adjusted by Pe a (Takara Io) to control the width of the genome. It was decided that there was no transfer of information.
  • the genome was extracted from the chicks of Transjik Watt (9), and 4 pieces of the amplified fragment by PC were used.
  • the plus with X (Zinc Access) selected was selected as C X.
  • the target piece was collected (Insert). SC eobac was cleaved with d 1, a e P os a ase P, purified and recovered. This was recovered as a gashose and the target piece (Kuta). The vector insert pieces were ligated and transformed into • co 5a a.
  • the blood samples were prepared from 796. All performed at 4C. White was diluted with 5 volumes of ⁇ 5 s C (P 6), ⁇ 5 a and suspended until the activity became low. Then, in step 2 of a, adjust to 7 and 4 and add CaC to the end. Centrifuge at 5X, collect, and pass through the 4.
  • Figure 8 shows the result of S S P G of the sump in the manufacturing step.
  • S S P G was carried out under the condition of 52 gents.
  • the three bands near 5 a 3 a 2 a, which are derived from the offspring, can be confirmed virtually.
  • the kinetics of human and offspring in rat fluid was examined. 5 was administrated, blood was collected over time, and the plasma was measured by the S method described in Separation 2. The reduction period is about 28.8 for human children,
  • the construction of the vector SC eobac obtained in. Indicates the column of whisking gna. There is a section of a in which the don (G) of a is changed to G in the quessing guna sequence of Usui's disease. Indicates a gene. Five
  • 25 shows the construction of the vector SC eobac e obtained in 25. Indicates the column of whisking gna.
  • the part of a that changes the don (G) of a to G is attached to the quessing guna sequence of the Uss disease. Indicates a gene. Five
  • WP is a regulator from Yak Wis.
  • trans-jik wat (7 9) serum and 11 white bloods which are trans-wat using the whistle vector and others are trans-economic wat using the SC eobace tow vector of implementation 5.
  • Mosquito is a molecular mosquito.
  • Novosen (Novonodisc) (human product) is a sump and a sump from the wild Transgenic Watts.
  • Child progenitor 5a cut into po When activated, it is 3 a 8 a.
  • Novosen is 2.
  • And plasma respectively, show the results of the gene transfer of the genomics of the trans-Wat (9) by 2 PC and 5 PC of electricity.
  • N is a molecular product
  • N is a PC product of the wild wat genome
  • N 2 is a PC product of the transgenic wat (9) genome
  • N 3 is a PC product of SC eobac.
  • the genome was 8 SC eobac 1, and 4 out of 5 of 5 cyclis were identified.
  • the target (97b) cannot be confirmed with, but the target 2 can be confirmed with target 2.
  • the construction of the X vector SC eobac X obtained in 6 is shown. Indicates the column of whisking gna. There is a section of a in which the don (G) of a is changed to G in the quessing guna sequence of Usu blood disease. X indicates the X gene. 5 3 indicates the SC column.
  • FIG. 7 shows the structure of the obtained protein C vector SC eobac o e C. Indicates the column of whisking gna. There is a section of a in which the don (G) of a is changed to G in the quessing guna sequence of Usui's disease. o e C indicates the protein C gene. 5 3 indicates the SC column.
  • Molecule 5a Human type molecule 3a2 Can be checked. Due to the manufacturing process, it is possible to almost clearly confirm the three bands near 5 a 3 a 2 a that are caused by the child.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Environmental Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L’invention concerne une nouvelle méthode pour produire une protéine ayant un résidu Gla. La méthode est caractérisée par la production d’une protéine ayant un résidu Gla au moyen d'un oiseau transgénique, ledit oiseau transgénique étant préparé en infectant par micro-injection le coeur ou un vaisseau sanguin formé dans un embryon d'oiseau à l'étape initiale du développement embryonnaire avec un vecteur de rétrovirus ayant un gène étranger, et en faisant éclore l'embryon.
PCT/JP2006/324593 2005-12-08 2006-12-08 OISEAUX TRANSGÉNIQUES AYANT UN RÉSIDU Gla DANS UNE PROTÉINE DÉRIVÉE D’UN GÈNE ÉTRANGER WO2007066775A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005355038 2005-12-08
JP2005-355038 2005-12-08

Publications (1)

Publication Number Publication Date
WO2007066775A1 true WO2007066775A1 (fr) 2007-06-14

Family

ID=38122920

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/324593 WO2007066775A1 (fr) 2005-12-08 2006-12-08 OISEAUX TRANSGÉNIQUES AYANT UN RÉSIDU Gla DANS UNE PROTÉINE DÉRIVÉE D’UN GÈNE ÉTRANGER

Country Status (1)

Country Link
WO (1) WO2007066775A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004000366A1 (fr) * 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Glycoformes du facteur vii pegylees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004000366A1 (fr) * 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Glycoformes du facteur vii pegylees

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KAMIHIRA M. ET AL.: "Transgenic Niwatori ni yoru Human Erythropoietin no Seisan (Production of human erythropoietin using transgenic chickens)", THE SOCIETY FOR BIOTECHNOLOGY, JAPAN TAIKAI KOEN YOSHISHU, vol. 2005, 25 September 2005 (2005-09-25), pages 214 (1H14-3, XP003013865 *
KWON M.S. ET AL.: "Development of transgenic chickens expressing enhanced green fluorescent protein", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 320, 2004, pages 442 - 448, XP004516650 *
SAKAI T. ET AL.: "The gamma-carboxyglutamic acid domain of human factor VIIa is essential for its interaction with cell surface tissue factor", J. BIOL. CHEM., vol. 265, no. 4, 1990, pages 1890 - 1894, XP002373510 *
UEHIRA M. ET AL.: "Retrovirus Vector ni yoru Transgenic Chorui Sakusei ni okeru Donyujiki no Kento", THE SOCIETY OF CHEMICAL ENGINEERS, JAPAN NENKAI KENKYU HAPPYO KOEN YOSHISHU, vol. 70, 22 February 2005 (2005-02-22), pages 325 (G315), XP003009867 *

Similar Documents

Publication Publication Date Title
JP7688682B2 (ja) 核酸分子およびその使用
EP1395293B1 (fr) Vecteurs lentiviraux codant des facteurs de coagulation utiles pour la therapie genique
AU2019357039B2 (en) Generation of improved human pah for treatment of severe PKU by liver-directed gene replacement therapy
US6825396B2 (en) Methods for tissue specific synthesis of protein in eggs of transgenic hens
JP7644007B2 (ja) 第ｉｘ因子を発現するレンチウイルスベクターの使用
US9974288B2 (en) Method of making polyethylene glycol-modified mammalian erythropoietin in a transgenic chicken
JP5198747B2 (ja) ネコ由来タンパク質のコード配列を含む外来性遺伝子を含むトランスジェニック鳥類およびその作製法
WO2006112147A1 (fr) Oiseau transgenique produisant de l’erythropoietine et procede pour l’obtenir
JP2015536148A (ja) 改変第ｉｘ因子タンパク質についての方法および組成物
WO2004016081A1 (fr) Technique d'expression de gene dans des oiseaux transgeniques au moyen de vecteur retroviral et oiseaux transgeniques ainsi obtenus
WO2007066775A1 (fr) OISEAUX TRANSGÉNIQUES AYANT UN RÉSIDU Gla DANS UNE PROTÉINE DÉRIVÉE D’UN GÈNE ÉTRANGER
JP5280001B2 (ja) 外来性遺伝子由来のタンパク質がＧｌａ残基を含むトランスジェニック鳥類及びその作製法
WO2009142186A1 (fr) Composition cytotoxique
WO2005021769A1 (fr) Procede de construction d'un oiseau transgenique a l'aide d'un vecteur lentivirus et oiseau transgenique obtenu
RU2816873C2 (ru) Применение лентивирусных векторов, экспрессрующих фактор ix
JP2008253158A (ja) クリングル構造を有するタンパク質の新規生産方法
JP2007275004A (ja) レンチウイルスベクターの精巣への感染による遺伝子組換え鳥類作製法
NZ503901A (en) Transgenic avians containing replication-defective retroviral vectors and methods for tissue specific synthesis of protein in eggs laid by the avian or female progeny thereof.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06834348

Country of ref document: EP

Kind code of ref document: A1