WO1997008947A1 - Transformant - Google Patents

Transformant Download PDF

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Publication number
WO1997008947A1
WO1997008947A1 PCT/JP1996/002402 JP9602402W WO9708947A1 WO 1997008947 A1 WO1997008947 A1 WO 1997008947A1 JP 9602402 W JP9602402 W JP 9602402W WO 9708947 A1 WO9708947 A1 WO 9708947A1
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WIPO (PCT)
Prior art keywords
thr
pro
val
ser
stem cells
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PCT/JP1996/002402
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English (en)
Japanese (ja)
Inventor
Takashi Okado
Yi Zhang
Hideyuki Matsushita
Kiyozo Asada
Ikunoshin Kato
Original Assignee
Takara Shuzo Co., Ltd.
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Application filed by Takara Shuzo Co., Ltd. filed Critical Takara Shuzo Co., Ltd.
Priority to AU68365/96A priority Critical patent/AU6836596A/en
Publication of WO1997008947A1 publication Critical patent/WO1997008947A1/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated

Definitions

  • the present invention relates to a transformant having excellent growth properties and a method for producing the same.
  • transgenic embryos that incorporate the foreign genomic material (DNA) into the nucleus of the embryo or infect it with the substance integrated into the chromosome of the embryo.
  • DNA foreign genomic material
  • This embryo can be grown by transplantation into a foster parent, and the resulting adult animal has incorporated the foreign DNA into its chromosome and can express it.
  • Transformed individuals are generally called transgenic animals [Science, vol. 214, pp. 1244-1246 (1981)].
  • the incorporated exogenous DNA is called a transgene and generally consists of a promoter and a gene of interest such as cDNA.
  • the transgenic animal Since the transgenic animal is produced as an animal having the target gene by transplanting the transformed embryo into a foster parent, developing and giving birth to the offspring, it takes a long time to produce the transgenic animal. In addition, when the probability of obtaining an animal having the target gene is low, a large number of mother animals as foster mothers are required.
  • An object of the present invention is to develop a method for transforming a living body without using a foster parent, and obtain a transformant capable of growing in a short period of time and the transformant. It is to provide a method for.
  • the first invention of the present invention relates to a growth transformant, comprising the following steps (A) and (B):
  • step (B) a step of introducing the stem cells obtained in the step (A) into a living body
  • the second invention of the present invention relates to a method for producing a growth transformant, comprising the following steps (A), (B) and (C):
  • step (B) a step of introducing the stem cells obtained in the step (A) into a living body
  • step (C) a step of expressing growth hormone in the living body obtained in the step (B).
  • a novel growth-transformed organism using a genetically modified living stem cell is provided, and the organism can be efficiently produced in a short period of time without using embryonic tissue, and thus without the need for a foster parent. It is a very useful organism that has excellent growth potential, grows in a short period of time, and has improved weight gain and milk yield.
  • the transformants of the present invention include mammals (eg, mice, rats, rabbits, sheep, sheep, goats, pigs, pigs, horses, dogs, monkeys, chimpanzees, etc.), birds (eg, chickens, turkeys). , Quail, ducks, ducks, etc.), reptiles (eg, snakes, turtles, etc.), amphibians (eg, power elephant, sanshowo, imimo etc.), fishes (eg, horse mackerel, mackerel, sea bass, sea bass, grouper It is an organism such as yellowtail, tuna, bonito, salmon, trout, perch, flounder, shark, ray, sturgeon, etc.).
  • mammals eg, mice, rats, rabbits, sheep, sheep, goats, pigs, pigs, horses, dogs, monkeys, chimpanzees, etc.
  • birds eg, chickens, turkeys.
  • a stem cell is prepared from the living organism of the organism, a gene encoding a growth hormone is incorporated into the stem cell, and then a recombinant stem cell incorporating the gene encoding the growth hormone is introduced into the living organism to obtain the stem cell.
  • the growth hormone in the present invention includes a growth hormone of the same species as the transformant and a heterologous growth hormone which does not show antigenicity in the transformant and can exert its action on the organism. It is a transformant that exhibits stable expression and function.
  • Mammalian growth hormone is produced in the pituitary gland, and their activity and structure are known. For example, J. Human Growth Hormone.
  • Gastrointestinal hormonal growth hormone gene JP-A-63-197
  • porcine growth hormone gene Japanese Unexamined Patent Publication (Kokai) No. 59-173083
  • human growth hormone gene Science, vol. 205, p. 602 (1979)] and the like
  • the growth hormone gene of avian two Wa tri growth hormone gene is known [J. Exp. 2 00 1. # 232, pp. 465].
  • the growth hormone gene of fish is also the growth hormone gene of salmon (JP-A-61-9).
  • the target growth hormone gene may be isolated by a known method and used. Further, a known isolated growth hormone gene may be used.
  • the growth hormone is not limited to natural growth hormone, but a polypeptide having substantially the same activity as these growth hormones, that is, a growth hormone which has essentially the same activity as natural growth hormone. It includes polypeptides in which the amino acid of growth hormone has been replaced with other amino acids, polypeptides in which amino acids have been deleted, and polypeptides in which amino acids have been inserted, without impairing them.
  • the living body stem cell in the present invention is a cell having a pluripotency derived from a living body after differentiation from embryonic tissue and having a self-renewal ability.
  • Examples thereof include hematopoietic stem cells, peripheral blood stem cells, epithelial stem cells, exocrine gland stem cells, and endocrine glands.
  • Stem cells hepatic stem cells, Teng stem cells, neuroendocrine stem cells, connective tissue stem cells, stromal stem cells, fibroblast stem cells, mesenchymal stem cells, adipocyte stem cells, breast gland stem cells, reticuloendothelial stem cells, lipid stem cells, chondrocytes Stem cells, osteoproge nitor stem cells, osteocyte stem cells, muscle fiber stem cells, neuronal stem cells, epidermal stem cells, keratinocyte stem cells, Langerhans stem cells, melanocyte stem cells, sebaceous stem cells, sweat gland stem cells, mucus stem cells , Serosal stem cells, odontogenic stem cells, islet stem cells, alveolar stem cells Cells, retinal stem cells and the like can be used.
  • stem cells can be obtained from various tissues of a living animal to be used.
  • the hematopoietic stem cells are cells not exist only at a frequency of 1 Bruno 1 0 5 in the bone marrow cells
  • stearyl ⁇ cell factor one cell surface receptor of the hematopoietic stem cell (stem cell factor: SCF) receptor It can be purified using an antibody against the c-kit receptor [Blood, Vol. 78, pp. 176-172 (1991)].
  • a method of introducing a growth hormone gene into stem cells calcium phosphate, microinjection, electroporation, adenovirus, SV40 virus, simple virus, etc. are used. A method using a retroviral vector is most suitable.
  • the gene of interest can be stably inserted into the chromosome of a dividing cell.
  • a replication-defective system has been established so that a recombinant virus does not produce a new virus even if it infects target cells, and a high-titer, stable supply method has been reported (Hum. Gen. Ther. Vol. 5, pp. 19-28 (1994)].
  • the infection efficiency depends on the titer of the recombinant virus, co-culture of recombinant virus-producing cells and target cells is necessary for highly efficient gene transfer into hematopoietic stem cells [Nature, No. 3 Vol. 10, p. 476 (1994)].
  • the fibronectin fragment is represented by SEQ ID NO: 10 in the sequence listing described in Japanese Patent Application Laid-Open No. 2-111498, and is Escherichia coli. ichia coli)
  • a polypeptide produced by HB101 / pHD102 (FERM P-10721), also represented by SEQ ID NO: 11 in the sequence listing, and Escherichia coli HB101 / pCH102 (FERMBP) -2800), and using these polypeptides, it is possible to introduce a gene encoding a heterologous protein into target cells with high efficiency by the above method.
  • FERM P-10721 also represented by SEQ ID NO: 11 in the sequence listing
  • Escherichia coli HB101 / pCH102 (FERMBP) -2800
  • the growth hormone gene integrated into a retroviral vector generally consists of a promoter and DNA encoding growth hormone.
  • various promoters that function in the organism can be used as the promoter.
  • mouse metallothionein I promoter-1 [Nature, vol. 296, p. 39 (1982)] and the like can be used. it can.
  • a promoter such as LTR provided in the retrovirus vector can also be used.
  • only the DNA encoding growth hormone need be inserted downstream of the promoter.
  • a cDNA prepared from the growth hormone mRNA or a growth hormone-encoding region DNA derived from genomic DNA can be used.
  • the recombinant stem cell into which the gene encoding the target growth hormone has been introduced is introduced into a living organism.
  • the stem cells may be administered intravenously, or may be directly injected into the tissue from which the stem cells are derived.
  • hematopoietic stem cells When hematopoietic stem cells are used as recombinant stem cells, living organisms may have a compromised immune function. As a method for deficient immunity of the living animal, for example, irradiation may be performed, and an immunocompromised drug may be used. May be administered.
  • a pure mouse is irradiated with about 1,000 rads of radioactivity. This dose is lethal to mice, after a while blood cells are depleted, white blood cells are lost, immunity is compromised, infection occurs, and death occurs in about two weeks [Rad. Res. Vol. 14, pp. 31-32 (1961)].
  • mice can continue to survive due to hematopoietic cells reconstituted from the administered hematopoietic stem cells.
  • immunodeficiency drugs include anti-lymphocyte serum, corticosteroids such as prednisolone, methylprednisolone, and dexamethasone; purine antagonists such as azathioprine and 6-MP; alkylating agents such as cyclophosphamide and busulfan; Cyclosporin 8, FK506 etc. can be used.
  • anti-lymphocyte serum administer intravenously or intramuscularly a dose of 1-2 Omg / kg as agropurine, and 10--15 when using methylprednisolone as a corticosteroid. High doses of mgZkg may be given.
  • azathioprine When azathioprine is used, 2-3 mgZkg may be administered, and when cyclophosphamide is used, a large dose of 40-5 Omg / kg may be administered.
  • these immunodeficiency drugs the immune ability of living animals is compromised. Similarly, an immunodeficient living animal can survive by introducing recombinant hematopoietic stem cells.
  • the target growth hormone gene is permanently expressed in the transgenic organism, and can transform a host organism. It is preferable to introduce the growth hormone gene into the living body during the period before growth, the introduced growth hormone gene is expressed in the living body, and the expressed growth hormone enhances skeletal growth, nitrogen retention, protein synthesis, and traits of the living body. Significant increase in growth rate, weight, and meat production in transformants Thus, a transformant of the present invention having excellent growth properties, that is, a transformant having a growth property can be obtained.
  • the present invention provides a transformed organism using the transgenic living stem cell.
  • the organism is very useful because it can be efficiently produced in a short period of time without using embryonic tissue, has excellent growth potential, and has an improved weight gain and milk yield.
  • mGH mouse growth hormone
  • mGH mouse growth hormone
  • a mouse brain cDNA library (Clontech) was used as type II DNA of PCR. Prepare 1 reaction solution of this cDNA library 1HF1, MGHF 1 and MGH R1 using 1 Opmol and Takara Shuzo PCR Amplification Kit, respectively, and prepare them at 94 ° C for 1 minute and at 55 ° C. Temperature cycles were shifted for 30 minutes at 72 ° C for 1 minute. After completion of the reaction, 51 was removed from the reaction solution and subjected to 1% agarose gel electrophoresis. As a result, a single band having a size of about 75 Obp was observed. This reaction solution was diluted 100-fold with water, the mixture was made into type II, and MGHF2 and MGHR2 were primed. When PCR and electrophoresis were performed under the same conditions as above, a single band of about 700 base pairs was observed, indicating that PCR was able to specifically amplify the desired mGH cDNA. .
  • a large amount of cDNA for ⁇ lGH was prepared by preparing 10 more reaction systems of the above 501 using the primers of MGHF1 and MGHR1 and performing PCR.
  • the cDNA fragment of about 75 Obp was replaced with TE buffer [1 OmM Tris-HCl ( pH 7.5) and 0.1 mM EDTA [50 xl]. Of these (20 Hi), the cDNA ends were blunted using the DNA Blunting Kit (Takara Shuzo), followed by phenol extraction and ethanol precipitation. The cDNA was recovered by centrifugation in 101 TE buffer, and a phosphate group was added to the 5 'end of cDNA using a MEGA LABEL kit (Takara Shuzo).
  • the plasmid vector pUC1180.2 (2 ul), which had been digested with the restriction enzyme Hincl I and then dephosphorylated with Calf Intestine Alkaline Phosphatase (Takara Shuzo), and the above-recovered cDNA fragment of about 0.5 / zg was mixed and the cDNA fragment was ligated to the vector DNA using DNA Ligation Kit Ver. 2 (Takara Shuzo).
  • E. coli J Ml09 was transformed using the reaction solution 4-1.
  • X-Gal and I An agar medium supplemented with PTG was used.
  • This linker is composed of a Pstl cleavage site BamHI site and a HindII site.
  • This linker 0.1 (0.05) was mixed with pMGH 7 Pstl and Hind III digest 0.5 fig (51), and pMGH was synthesized using the DNA Ligation Kit Ver. 2 described above. A linker sequence was inserted between the Pstl site of 7 and the Hind III site.
  • E. coli J M109 is transformed using reaction mixture 2 // 1, cultured on L-agar medium containing 100 gZml of ampicillin, and ampicillin resistant on the medium. Colonies were obtained. Then, 10 clones were arbitrarily selected, and each clone was cultured overnight in 5 ml of L-liquid medium containing 50 gZml of ampicillin, and a plasmid was prepared from 1.5 ml of the culture solution. . The restriction enzyme BamHI was allowed to act on the plasmid, and the digest was analyzed by 1% agarose gel electrophoresis. As a result, a fragment of about 75 Obp was confirmed.
  • the cDNA fragment of raGH excised with BamHI described above was prepared by extracting from the agarose gel, and the same procedure was carried out using the above ligation kit to carry out the same operation to obtain the retrovirus vector pZI.
  • P-NeoS V (X) 1 was inserted into the BamHI site of Cell. Vol. 37, p. 1053 (1984).
  • E. coli JMl09 was transformed with the reaction mixture 2 ⁇ 1, and cultured on an L-agar medium containing 50 zgZnil of ampicillin, and ampicillin-resistant colonies were cultured on the medium.
  • PCR was performed on 24 ampicillin-resistant colonies obtained on the obtained c- plate in combination with MGH F1 and the primers shown in SEQ ID NOs: 8 and 9 in the sequence listing to obtain the mGH cDNA in the correct direction.
  • a search for clones with the inserted fragments revealed that the four clones were the target.
  • the correct orientation means that the 5 'position of the sequence encoding the mGH cDNA is inserted into the UTR sequence of the LTR, and the 3' position is inserted into the neomycin resistance gene.
  • plasmid was named pRMGH3, and used in subsequent experiments.
  • Producer cells that produce a retrovirus incorporating the mGH cDNA sequence can be obtained from the above-described pRMGH3 by packaging mouse GP + E-86 derived from mouse NI HZ3T3 cells (AT CC CRL-1658) [J. Viol., 62, 1120 (1988)] by the calcium phosphate method. The transfection was performed according to the protocol described in the laboratory manual Animal Genetic Engineering [Michael Kriegler, translated by Ikuyuki Kato, Takara Shuzo Publishing Co., Ltd. (1994)], pp. 203-204.
  • the mGH cDNA sequence was integrated into the cell genome.
  • the titer of the virus in the cell supernatant produced from the seven clones was determined by the method described in the aforementioned Genetic Engineering of Animal Cells, pp. 204-206. Producer cells showing the highest titer (2 ⁇ 1 OsCFUZnil) were used for subsequent gene transfer experiments into hematopoietic stem cells.
  • Primers MMETF1, MMETF2, and MMETR2 for obtaining a DNA fragment containing the mouse metamouth thionein I promoter (hereinafter abbreviated as mMET promoter) are described in Nature, Vol. 292, p. 267 (1981), and Nature, respectively. Designed with reference to the nucleotide sequence described in Vol. 296, p. 39 (1982) did.
  • the nucleotide sequences of primers MMETF1, MMETF2, and MMETR2 are shown in SEQ ID NOs: 12, 13 and 14, respectively, in the sequence listing.
  • a part of the reaction solution was subjected to agarose gel electrophoresis to amplify a DNA fragment corresponding to about 42 Obp.Acn, Bgin, Haen, HapE, Sac I (all manufactured by Takara Shuzo) After confirming that the fragment contained the mM ET promoter region by restriction enzyme digestion using), amplified DNA fragments were recovered from the reaction solution using the QI Aquick PCR Purification Kit (Qiagen). .
  • the nucleotide sequence of the DNA fragment was determined by the didequin method. Two of the three plasmids contained a region of the mMET promoter having the same nucleotide sequence as in the above-mentioned literature, and one of them was named plasmid pMPT and used for the subsequent experiments.
  • the nucleotide sequence of the PCR-amplified DNA fragment inserted into plasmid pMPT is shown as SEQ ID NO: 15 in the sequence listing.
  • Plasmid pHGHG containing genomic DNA encoding human growth hormone (hereinafter abbreviated as hGH) was obtained from Nicholas Institute Diagnostic.
  • the nucleotide sequence of an approximately 2.1 kb BamHI-EcoRI DNA fragment containing the hGH coding region in the plasmid is shown in SEQ ID NO: 16 in the sequence listing.
  • the nucleotide sequences of primers HGHF4 and HGHR4, which were designed based on the base sequence and were used in the detection of the DNA fragment containing the hGH coding region in the following examples, are shown in SEQ ID NO: 17 and SEQ ID NO: 17, respectively. See Figure 18.
  • the DNA fragment contains the genomic DNA encoding the mMET promoter and hGH located downstream thereof, and has BamHI ends at both ends.
  • the pEmi vector which is a retrovirus vector, is composed of a 2.2 kb SalI-Clal fragment derived from a pLRNL vector [Virology ⁇ Vol. 171: 331 (1989)] and a pBabe Blue vector [Nucl. Acids. Res. Vol. 35, p. 87 (1990)].
  • Plasmid pMHE is a retrovirus vector containing genomic DNA encoding hGH located downstream of the raMET promoter and a neomycin resistance gene as a selectable marker.
  • NI HZ3T3 cells were modified from dalbecco containing 10% fetal calf serum (FCS, manufactured by Dainippon Pharmaceuticals) and 100 units / ml of penicillin and 1 ⁇ 0 gZml of streptomycin (both manufactured by Gibco). After culturing in Eagle's medium (DMEM, manufactured by JRH Biosciences), and adding 70 g of confluent, add 10 g of plasmid pMHE together with DOTAP transfection solution (Boehringer Mannheim), and add cells. Transfection was performed.
  • FCS fetal calf serum
  • FCS fetal calf serum
  • streptomycin both manufactured by Gibco
  • DMEM All the media hereinafter referred to as DMEM contain FCS, benicillin, and streptomycin as described above.
  • G418-resistant cells were selected according to the method described in Yodosha, 1994, Gene Transfer and Expression, Analytical Methods, pp. 74-80.
  • the obtained G418-resistant colonies were transferred to a new dish, and cultured in the above medium containing 0.7 SingZml of G418 at a final concentration of 90% confluent.
  • the culture ground in the above medium containing Z n S 0 4 at a final concentration of 100 M, and incubated an additional 4 0 h.
  • the culture supernatant was collected and filtered through a 0.22 m filter (Millipore), and 20/1 was subjected to 15% SDS-PAGE. Was.
  • the gel was subjected to Western blotting using an anti-human growth hormone antibody (UCB Bioproducts) according to the method described in Ryrent 'Protocol in Molecular' Technology, 10.2.2 to 10.2.10. Was done.
  • UMB Bioproducts anti-human growth hormone antibody
  • 1 zg of purified human growth hormone manufactured by Seikagaku Corporation was used. As a result, it was confirmed that hGH reacting with the above antibody was generated in the culture supernatant.
  • Producer cells that produce recombinant retroviruses for use in the introduction of the hGH gene are packaging cells that contain the above plasmid pMHE. + £ -86 prepared by transfection. Transfection of the cells was performed in the same manner as in the transfection of NIHZ3T3 cells described above. From the obtained G418-resistant colonies, 50 colonies distant from other colonies were selected, and were simply obtained by the method described in REALIZE INC., Animal Cell Practical Use Manual, pages 88 to 89 (1984). Released. After culturing the isolated cells to 90-95% confluence in a 90-dragon dish, genomic DNA was prepared from the cells collected by centrifugation in the manner described above.
  • genomic DNA was synthesized by PCR using primers HGHF4 and NEOR1 (25-cycle reaction with 94 ° C, 1 minute to 55 ° C, 1 minute to 72 ° C, 2 minutes as one cycle) was performed. Then, cells in which amplification of a DNA fragment of about 1.9 kb was observed were selected. The plasmid pMHE DNA is integrated on the genomic DNA of these cells.
  • the virus producing ability of the obtained producer cells was evaluated as follows. After these cells were grown in a semi-confluent in a 90-band dish, 9 ml of DMEM was added to the dish, cultured overnight, centrifuged, and the supernatant was collected. Filter the collected culture supernatant with a 0.45 ⁇ m filter ( (Lipore), to give a virus supernatant. The virus titer in these virus supernatants was measured using NI HZ3T3 cells by a standard method [J. Virol., Vol. 62, pp. 1120-1124 (1988)].
  • DMEM containing 2000 NIH / 3T3 cells per 1-well was added to a 6-well tissue culture plate, cultured overnight, and the serially diluted virus supernatant was added to a final concentration of 7.5 / g.
  • the mixture was added to each well together with 1 / ml hexadimetrim-promide (Polybrene: Aldrich). After incubating the plate at 37 ° C for 24 hours, the medium was exchanged for one containing G418 (final concentration: 0.75 mg / ml, Gibco), and the plate was further incubated.
  • mice Six-week-old mice (C 3HZH e J) were administered 15-mg / kg of 5-fluorouracil (5-FU, manufactured by Amresco) via the tail vein, and two days later, the femur and ribs were removed and bone marrow was removed. Collected. The obtained bone marrow was subjected to density gradient centrifugation using Ficoll-Hypaque (density 1.0875 gZml, manufactured by Pharmacia) to prepare a low-density mononuclear cell fraction, which was used as mouse bone marrow cells. Mouse bone marrow cells were prestimulated prior to retrovirus infection according to the method of Lusky et al. [Blood, Vol. 80, p.
  • the above mouse bone marrow cells were co-cultured with the producer cells prepared in Example 1-1 (2) or Example 2- (3) in the presence of rhlL-6 and rrSCF at 37 ° C for 24 hours. .
  • the medium used for the above pre-stimulation of bone marrow cells was used, and 7.5 g / ffll of polypropylene was further added. After the culture, non-adherent cells were collected and used as transgenic bone marrow cells.
  • transgenic bone marrow cells described above were administered from the tail vein of a 6-week-old C3HZHe J mouse, and three days later, the transgenic bone marrow cells were administered again.
  • mice to which physiological saline was administered instead of bone marrow cells were prepared and bred in the same manner.
  • mice to which bone marrow cells transfected with the producer cells prepared in Example 2 (3) having the mMET promoter were administered, 25 mM ZnS Z Given water containing 4 .
  • mice receiving the bone marrow cells transfected with the growth hormone gene were significantly more weighed than the control group (mean 25 g). There was an increase. Furthermore, growth hormone gene is under the control of mMET promoter, in the group given water containing Z n S 0 4 of 25 mM, further weight gain was observed.
  • the efficiency is improved without using a foster parent.
  • Good growth transformants can be provided.
  • the transformant has excellent growth properties, and can permanently enlarge a living body by expressing growth hormone permanently.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Ala lie Pro Ala Pro Thr Asp Leu Lys Phe Thr Gin Val Thr Pro
  • Lys Glu lie Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser
  • Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
  • 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe
  • Asp Tyr Lys lie Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type other nucleic acid (PCR amplified DNA fragment)
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)

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Abstract

Cette invention concerne un organisme qui est transformé de manière efficace et sans faire appel à un hôte grâce à un processus comprenant les étapes suivantes: (A) intégrer un gène codant une hormone de croissance dans des cellules souches vitales, et (B) introduire dans l'organisme les cellules souches obtenues lors de l'étape (A).
PCT/JP1996/002402 1995-09-08 1996-08-28 Transformant WO1997008947A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002511094A (ja) * 1997-07-14 2002-04-09 オシリス セラピューティクス，インコーポレイテッド 間葉幹細胞を用いる心筋再生
WO2015034714A1 (fr) * 2013-09-06 2015-03-12 3M Innovative Properties Company Bande de marquage de chaussée avec couche de finition de polyuréthane modifié

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JP2002511094A (ja) * 1997-07-14 2002-04-09 オシリス セラピューティクス，インコーポレイテッド 間葉幹細胞を用いる心筋再生
WO2015034714A1 (fr) * 2013-09-06 2015-03-12 3M Innovative Properties Company Bande de marquage de chaussée avec couche de finition de polyuréthane modifié
US10344133B2 (en) 2013-09-06 2019-07-09 3M Innovative Properties Company Pavement marking tape with modified polyurethane topcoat

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