WO1997008947A1 - Transformant - Google Patents

Abstract

An organism is efficiently transformed without using any host by a process involving the step (A) of integrating a gene encoding a growth hormone into vital stem cells and the step (B) of introducing the stem cells obtained in the step (A) into the organism.

Description

 Description Transformant Field of the Invention

 The present invention relates to a transformant having excellent growth properties and a method for producing the same.

 Conventional technology

 Recent developments in developmental engineering have made it possible to create 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. [Pro Natl. Acad. Sci., USA, Vol. 77, pp. 7380-7384 (1980); Cell, Vol. 32, pp. 209-216 (1982)]. 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.

 Purpose of the invention

 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.

 Summary of the Invention

 The first invention of the present invention relates to a growth transformant, comprising the following steps (A) and (B):

 (A) incorporating a gene encoding growth hormone into living stem cells, and

 (B) a step of introducing the stem cells obtained in the step (A) into a living body,

It is a novel growth transformant obtained by the step comprising:

 The second invention of the present invention relates to a method for producing a growth transformant, comprising the following steps (A), (B) and (C):

 (A) a step of incorporating a gene encoding growth hormone into living stem cells,

(B) a step of introducing the stem cells obtained in the step (A) into a living body, and

(C) a step of expressing growth hormone in the living body obtained in the step (B).

 According to the present invention, 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.

 Detailed description of the invention

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.). 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. be able to. 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.

Am. Cheni. Soc. 80, 4429 (1958), Biochem. J. 100, 754 (1966), etc. For the growth hormones of fish, examples of isolation from tilapia [Gen. Comp. Endocrin., Vol. 30, p. 91 (1976)], examples of isolation from sturgeon [Endocrinology II, Vol. 108, p. 377] (1981)], and examples of isolation from koi [Gen. Comp. Endocrin., Vol. 50, p. 335 (1983)]. For the growth hormone gene in mammals, the rat growth hormone gene [Nature, vol. 270, p. 486 (1977)], the mouse growth hormone gene [J. Biol. Chem., Vol. 260, p. 9574] (1985)], Gastrointestinal and porcine growth hormone genes [DNA, Vol. 2, p. 37 (1983)]., 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 are known. 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). Japanese Patent Application Laid-Open Nos. 1991-1991 and 61-210100, Red Sea Bream Growth Hormone Gene (Japanese Unexamined Patent Application Publication No. Hei 2-219576), Japanese Flounder Growth Hormone Gene — 238 84), tuna growth hormone gene (Japanese Patent Application Laid-Open No. 11-180180), katso growth hormone gene (Japanese Patent Application Laid-Open No. 1-165085), and the like. Separated.

 In the present invention, the target growth hormone gene may be isolated by a known method and used. Further, a known isolated growth hormone gene may be used. In the present invention, 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. These stem cells can be obtained from various tissues of a living animal to be used. For example, although the hematopoietic stem cells are cells not exist only at a frequency of 1 Bruno 1 0 ⁵ in the bone marrow cells, stearyl厶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)].

 As 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.

 For example, if a retrovirus vector is used, 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)]. Although 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)].

 On the other hand, as a method for adding the virus supernatant, that is, only the virus particles to the target cells, culturing, and introducing the gene with high efficiency, for example, in a culture dish coated with a fibronectin (FN) fragment, By culturing the target cells and the target cells, it is possible to introduce the target gene with the same high efficiency as in co-culture [J. Clin. Invest., Vol. 93, pp. 1445-1 (19 9 4)].

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. Such a method is described in WO 95/26200 published October 5, 1995.

 The growth hormone gene integrated into a retroviral vector generally consists of a promoter and DNA encoding growth hormone. In that case, various promoters that function in the organism can be used as the promoter. For example, mouse metallothionein I promoter-1 [Nature, vol. 296, p. 39 (1982)] and the like can be used. it can. Further, a promoter such as LTR provided in the retrovirus vector can also be used. In this case, only the DNA encoding growth hormone need be inserted downstream of the promoter. As the growth hormone-encoding DNA, 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. As a method for introducing stem cells 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.

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.

 For example, 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)]. When recombinant hematopoietic stem cells are administered to lethal radiation-irradiated mice, mice can continue to survive due to hematopoietic cells reconstituted from the administered hematopoietic stem cells.

 Examples of 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. When using 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. 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. By using 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.

 Thus, 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.

 Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

 Example 1

 (1) Construction of retroviral vector PRMGH3 incorporating mouse growth hormone gene

 CDNA encoding mouse growth hormone (hereinafter abbreviated as mGH) was prepared by the PCR method.

 Based on the mouse growth hormone (mGH) cDNA sequence described in J. Biol. Chem. Above, four primers, MGHF1, MGHF2, MGHR1, and MGHR2, were selected. The sequences of each primer are shown in SEQ ID NOs: 1 to 4 in the sequence listing, respectively.

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 πlGH was prepared by preparing 10 more reaction systems of the above 501 using the primers of MGHF1 and MGHR1 and performing PCR.

 Then, after removing free primers, dNTPs and salts contained in the buffer with SUPREC-02 (manufactured by Takara Shuzo Co., Ltd.), 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). However, not labeled, but unlabeled ATP (1 OmM) was used here. After phenol extraction and ethanol precipitation, cDNA was recovered in 10 / il TE buffer, and 1〃1 of the cDNA was subjected to 1% agarose gel electrophoresis to check the amount of recovered cDNA.

Then, 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). After completion of the ligation reaction, E. coli J Ml09 was transformed using the reaction solution 4-1. Here, in order to select the clone into which the cDNA was inserted, X-Gal and I An agar medium supplemented with PTG was used. Arbitrarily selected 10 white colonies clearly distinguishable from the blue colony containing no insert, using the hot extract of the colony cells as type III, and using MGHF 1 and MGHR 1 as primers. When PCR was performed under the above conditions, an amplified band of about 75 Obp was observed in 8 clones. After preparing plasmid DNA from each clone, the nucleotide sequence of the inserted cDNA fragment was determined by the Dideoxy Chain Termination method, and had the same nucleotide sequence as that described in J. Biol. Chem. A clone having the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing was named PMGH7 and used in subsequent experiments.

 Synthesize oligodeoxyribonucleotides with a phosphate group added to the 5'-position shown in SEQ ID NO: 6 and SEQ ID NO: 7 in the sequence listing, and put them in TE buffer at a concentration of 1 zgZzl. After mixing and dissolving, the mixture was treated at 75 ° C. for 1 minute, and then gradually cooled to room temperature to anneal both oligodeokine ribonucleotides to prepare a linker. 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.

After completion of the ligation reaction, 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. In these clones, the linker sequence was inserted, and as a result, it was clarified that the cDNA fragment of mGH was cut out with BamHI. One clone was arbitrarily selected from these eight clones, and the plasmid was named pMGH12.

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). After completion of the ligation reaction, 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.

 Here, 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.

 One clone was arbitrarily selected from the four clones, the plasmid was named pRMGH3, and used in subsequent experiments.

 (2) Preparation of producer-cell

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. Then, on a selective medium containing G418 at a concentration of 800 gZinl, randomly select 15 colonies formed at positions well separated from other colonies, published by REALIZE INC, edited by Keiko Asada, and a manual for animal cell practical use Pp. 88-89 (1984), and cells of each clone were cultured separately in 90 band dishes until they reached 80% confluence. The cells were collected by centrifugation to prepare genomic DNA, 1 g of which was used as type III, and the primers MGHF1 and MGHR1 used in Example 11- (1) were subjected to PCR under the same conditions as in Example 11- (1). As a result, an amplified band of about 750 bp was confirmed in 7 clones. This means that in these seven clones, 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.

 Example 2

 (1) Acquisition of mouse metaoral thionein I promoter

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.

 Mouse genomic DNA was obtained from NI HZ3 T3 cells (ATCC CRL-1658), published by John Wiley & Sons Inc., 1987, Current Protocols in Molecular Biology. , 2.2.1 to 2.2.3.

 Using the PCR DNA Amplification Kit (Takara Shuzo), prepare 501 PCR reactions containing 250 ng of the above genomic DNA and 1 Opmol of each of the primers MMETF1 and MMETR2, and prepare them at 94 ° C for 1 minute. After the heat treatment, 30 cycles of 94, 1 minute to 56 ° C, 1 minute to 72 ° C, and 1 minute as one cycle were performed. 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). .

Of the recovered 42 ObpDNA fragments, 15 Ong was mixed with 5 Ong of pT7Blue T vector (Novagen) and ligated using DNΑ Ligation Kit V er. 2 (Takara Shuzo). After that, E. coli JM109 was transformed using the-part of the reaction solution. Some of the resulting transformation colonies were selected, and PCR was performed using MMETF2 and T7 promoter primers (Stratagene) (94 ° C, 30 seconds to 55 ° C, 30 seconds to 72 ° C, 30 A colony containing the target DNA fragment was searched for by a 25-cycle reaction with one cycle as one second). Three of the colonies thus selected were cultured to prepare a plasmid, which was inserted into the plasmid. 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.

 (2) Construction of retroviral vector pMHB incorporating human growth hormone gene

 After digesting the above plasmid pMPT 2 ^ g with Kpnl and BglH (both from Takara Shuzo), agarose gel electrophoresis was performed, and a DNA fragment corresponding to about 370 bp including the mMET promoter region was recovered from the gel.

 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.

 After digestion of 5 g of the plasmid pHGHG with BamH I and EcoRI (both from Takara Shuzo), agarose gel electrophoresis was performed, and a DNA fragment of about 2. 1 kb containing a region encoding hGH was recovered from the gel.

After digesting 2 / zg of the plasmid vector pBluescriptn (KS-) (Stratagene) with Kpnl and EcoRI, agarose gel electrophoresis was performed, and a DNA fragment corresponding to about 3 kb was recovered from the gel. Of this, 12.5 ng was mixed with 50 ng of the approximately 2 kb DNA fragment containing the above hGH coding region and 2 ng of the approximately 370b _P DNA fragment containing the above mMET promoter region, After ligation was performed using DNA Ligation Kit Ver.2, E. coli JMl09 was transformed using a part of the reaction solution. Select some of the obtained transformant colonies and perform PCR using primers MMETF2 and HGHR4 (94 ° (: 1 minute to 55, 1 minute to 72 ° (: 1 minute, 1 cycle) A colony containing the desired DNA fragment was searched for by using this PCR. A colony in which amplification of a DNA fragment of about 1 kb was recognized by this PCR was selected, and a plasmid was prepared from the colony. De pMHB.

 After digesting 3 g of the plasmid pMHB with BamHI and Vspl (Takara Shuzo), agarose gel electrophoresis was performed, and a DNA fragment corresponding to about 2.4 kb was recovered from the gel. 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 ゝ Vol. 171: 331 (1989)] and a pBabe Blue vector [Nucl. Acids. Res. Vol. 35, p. 87 (1990)].

7. Digest 2 μg of pEmi vector with BamHI, dephosphorylate with alkaline phosphatase (CIAP, manufactured by Takara Shuzo), and perform agarose gel electrophoresis. The DNA fragment was recovered from the gel. Of these, 5 Ong was mixed with the above about 2.4 kb DNA fragment 20 Ong, ligated using the DNA Ligation Kit Ver. 2, and then partially digested with a part of the reaction solution. E. coli JM109 was transformed. Some of the resulting transformant colonies were selected, and primers HGHF4 and a primer designed based on the nucleotide sequence of the neomycin resistance gene on the pEmi vector were used. PCR method using mer NEOR 1 (SEQ ID NO: 19 in the Sequence Listing shows the nucleotide sequence of the primer NEOR) (94 ° C, 1 minute to 55, 1 minute to 72, and 2 minutes as one cycle) A colony containing the desired DNA fragment was searched for by a 25-cycle reaction). A colony in which amplification of a DNA fragment of about 1.9 kb was confirmed by this PCR was selected, and a plasmid was prepared from the colony and named plasmid pMHE. 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.

The hGH expression ability of the hGH gene on Brasmid pMHE was confirmed by the following procedure. That is, 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 ン 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. All the media hereinafter referred to as DMEM contain FCS, benicillin, and streptomycin as described above. After further culturing the cells in the above medium, 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. Followed by replacing 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. As a positive control, 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.

 (3) Preparation of producer cells

 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. Using this genomic DNA as type III, 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 μm 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)]. That is, 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. G418 resistant colonies that grew 10 to 12 days later were stained with crystal violet and the number was recorded.よ り From the value obtained by multiplying the number of colonies per well by the dilution ratio of the virus supernatant, calculate the number of infectious particles (cfuZml) contained in 1 ml of the supernatant, and use this as the titer of the supernatant. Was. Among the obtained cells, those having the highest titer of the supernatant were selected and used in the following procedures.

 Example 3

 (1) Preparation of mouse bone marrow cells

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. 396 (1992)]. That is, 20% FCS, 100 units Zml recombinant human interleukin-1 6 (r hi L-6, Amjiyun), 1 OOngZml recombinant rat stem cell factor [] rSCF, Pepco Tech, 50 units / ml penicillin and 50 z / gZml containing streptomycin - MEM was added (Gibco) above mouse bone marrow cells at a cell density of 1 X 10 ⁶ cells / ml in, in 5% C0 _2, for 48 hours at 37 ° C. Prestimulated cells, including those attached to the container, were aspirated and collected using pipets.

 (2) Generation of transgenic mice

 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. . For the culture, 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.

The 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. As a control, mice to which physiological saline was administered instead of bone marrow cells were prepared and bred in the same manner. After the administration, 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 .

Three weeks after the administration of the transgenic bone marrow cells, the body weight of the mice was measured, and as a result, the 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.

As described above, according to the present invention, 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 Listing SEQ ID NO: 1

Array length: 20

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array:

 AGCATCCTAG AGTCCAGATT 20

SEQ ID NO: 2

Array length: 20

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array:

 TTCCAAACTG CTCAGAGTCC 20 SEQ ID NO: 3

Array length: 17

Sequence type: nucleic acid

Number of chains: 1

Topology: linear Sequence type: synthetic DNA

Array:

 GGGGCAGGGA GGCACAG 17 SEQ ID NO: 4

Array length: 20

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array:

 ACAGGAGAGT GCAGCAGAGA 20 SEQ ID NO: 5

Array length: 758

Sequence type: nucleic acid

Number of chains: 2

 Topology: linear

Sequence type: cD N A

Array:

AGCATCCTAG AGTCCAGATT CCAAACTGCT CAGAGTCCTG TGGACAGATC ACTGCTTGGC 60 AATGGCTACA GACTCTCGGA CCTCCTGGCT CCTGACCGTC AGCCTGCTCT GCCTGCTCTG 120 GCCTCAGGAG GCTAGTGCTT TTCCCGCCAT GCCCTTGTCC AGTCTGTTTT CTAATGCTGT 180 GCTCCGAGCC CAGCACCTGC ACCAGCTGGC TGCTGACACC TACAAAGAGT TCGAGCGTGC 240 CTACATTCCC GAGGGACAGC GCTATTCCAT TCAGAATGCC CAGGCTGCTT TCTGCTTCTC 300 AGAGACCATC CCGGCCCCCA CAGGCAAGGA GGAGGCCCAG CAGAGAACCG ACATGGAATT 360

GCTTCGCTTC TCGCTGCTGC TCATCCAGTC ATGGCTGGGG CCCGTGCAGT TCCTCAGCAG 420

GATTTTCACC AACAGCCTGA TGTTCGGCAC CTCGGACCGT GTCTATGAGA AACTGAAGGA 480

CCTGGAAGAG GGCATCCAGG CTCTGATGCA GGAGCTGGAA GATGGCAGCC CCCGTGTTGG 540

GCAGATCCTC AAGCAAACCT ATGACAAGTT TGACGCCAAC ATGCGCAGCG ACGACGCGCT 600

GCTCAAAAAC TATGGGCTGC TCTCCTGCTT CAAGAAGGAC CTGCACAAAG CGGAGACCTA 660

CCTGCGGGTC ATGAAGTGTC GCCGCTTTGT GGAAAGCAGC TGTGCCTTCT AGCCACTCAC 720

CAGTGTCTCT GCTGCACTCT CCTGTGCCTC CCTGCCCC 758 SEQ ID NO: 6

Array length: 23

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array:

 GGGATCCGTC ATCTTGTCTT CCA 23 SEQ ID NO: 7

Array length: 31

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array: AGCTTGGAAG ACAAGATGAC GGATCCCTGC A 31 SEQ ID NO: 8

Array length: 18

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array:

 GGTAGCCGGA TCAAGCGT 18 SEQ ID NO: 9

Array length: 18

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Synthetic DNA

Array:

 AACACGGCGG CATCAGAG 18 SEQ ID NO: 10

Sequence length: 296

Sequence type: amino acid

Number of chains: 1 strand

Topology: linear Sequence type: peptide

Array:

 Ala lie Pro Ala Pro Thr Asp Leu Lys Phe Thr Gin Val Thr Pro

1 5 10 15

Thr Ser Leu Ser Ala Gin Trp Thr Pro Pro Asn Val Gin Leu Thr

 20 25 30

Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met

 35 40 45

Lys Glu lie Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser

 50 55 60

Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu

 65 70 75

Lys Asp Thr Leu Thr Ser Arg Pro Ala Gin Gly Val Val Thr Thr

 80 85 90

Leu Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala

 95 100 105

Thr Glu Thr Thr lie Thr lie Ser Trp Arg Thr Lys Thr Glu Thr

 110 115 120 lie Thr Gly Phe Gin Val Asp Ala Val Pro Ala Asn Gly Gin Thr

 125 130 135

Pro lie Gin Arg Thr lie Lys Pro Asp Val Arg Ser Tyr Thr lie

 140 145 150

Thr Gly Leu Gin Pro Gly Thr Asp Tyr Lys lie Tyr Leu Tyr Thr

 155 160 165

Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val lie Asp Ala Ser 170 175 180

Thr Ala lie Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr

 185 190 195

Pro Asn Ser Leu Leu Val Ser Trp Gin Pro Pro Arg Ala Arg lie

 200 205 210

Thr Gly Tyr lie lie Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg

 215 220 225

Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr lie

 230 235 240

Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr lie Tyr Val lie Ala

 245 250 255

Leu Lys Asn Asn Gin Lys Ser Glu Pro Leu lie Gly Arg Lys Lys

 260 265 270

Thr Asp Glu Leu Pro Gin Leu Val Thr Leu Pro His Pro Asn Leu

 275 280 285

His Gly Pro Glu lie Leu Asp Val Pro Ser Thr

 290 295 SEQ ID NO: 11

Array length: 574

Sequence type: amino acid

Number of chains: 1 strand

 Topology: linear

Sequence type: peptide

Array: Pro Thr Asp Leu Arg Phe Thr Asn lie Gly Pro Asp Thr Met Arg

1 5 10 15

Val Thr Trp Ala Pro Pro Pro Ser lie Asp Leu Thr Asn Phe Leu

 20 25 30

Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu

 35 40 45

Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu

 50 55 60

Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gin

 65 70 75

His Glu Ser Thr Pro Leu Arg Gly Arg Gin Lys Thr Gly Leu Asp

 80 85 90

Ser Pro Thr Gly lie Asp Phe Ser Asp lie Thr Ala Asn Ser Phe

 95 100 105

Thr Val His Trp lie Ala Pro Arg Ala Thr lie Thr Gly Tyr Arg

 110 115 120 lie Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp

 125 130 135

Arg Val Pro His Ser Arg Asn Ser lie Thr Leu Thr, Asn Leu Thr

 140 145 150

Pro Gly Thr Glu Tyr Val Val Ser lie Val Ala Leu Asn Gly Arg

 155 160 165

Glu Glu Ser Pro Leu Leu lie Gly Gin Gin Ser Thr Val Ser Asp

 170 175 180

Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu 185 190 195 Leu lie Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg

 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe

 215 220 225

Thr Val Pro Gly Ser Lys Ser Thr Ala Thr lie Ser Gly Leu Lys

 230 235 240

Pro Gly Val Asp Tyr Thr lie Thr Val Tyr Ala Val Thr Gly Arg

 245 250 255

Gly Asp Ser Pro Ala Ser Ser Lys Pro lie Ser lie Asn Tyr Arg

 260 265 270

Thr Glu He Asp Lys Pro Ser Met Ala lie Pro Ala Pro Thr Asp

 275 280 285

Leu Lys Phe Thr Gin Val Thr Pro Thr Ser Leu Ser Ala Gin Trp

 290 295 300

Thr Pro Pro Asn Val Gin Leu Thr Gly Tyr Arg Val Arg Val Thr

 305 310 315

Pro Lys Glu Lys Thr Gly Pro Met Lys Glu lie Asn Leu Ala Pro

 320 325 330

Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys

 335 340 345

Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg

 350 355 360

Pro Ala Gin Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro

365 370 375 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr lie Thr lie

380 385 390

Ser Trp Arg Thr Lys Thr Glu Thr lie Thr Gly Phe Gin Val Asp

 395 400 405

Ala Val Pro Ala Asn Gly Gin Thr Pro lie Gin Arg Thr lie Lys

 410 415 420

Pro Asp Val Arg Ser Tyr Thr lie Thr Gly Leu Gin Pro Gly Thr

 425 430 435

Asp Tyr Lys lie Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser

 440 445 450

Ser Pro Val Val lie Asp Ala Ser Thr Ala lie Asp Ala Pro Ser

 455 460 465

Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser

 470 475 480

Trp Gin Pro Pro Arg Ala Arg lie Thr Gly Tyr lie lie Lys Tyr

 485 490 495

Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg

 500 505 510

Pro Gly Val Thr Glu Ala Thr lie Thr Gly Leu Glu Pro Gly Thr

 515 520 525

Glu Tyr Thr lie Tyr Val lie Ala Leu Lys Asn Asn Gin Lys Ser

 530 535 540

Glu Pro Leu lie Gly Arg Lys Lys Thr Asp Glu Leu Pro Gin Leu

 545 550 555

Val Thr Leu Pro His Pro Asn leu His Gly Pro Glu lie Leu Asp 560 565 570

Val Pro Ser Thr SEQ ID NO: 12

 Array Length: 19

 Sequence type: nucleic acid

 Number of chains: 1

 Topology: linear

 Sequence type: Other nucleic acids (synthetic DNA)

 Array:

 TCTCGTAAAC TCCAGAGCA 19 SEQ ID NO: 13

Array length: 18

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array:

 GAGCCAGTCG TGCCAAAG 18 SEQ ID NO: 14

Array length: 18

Sequence type: nucleic acid

Number of chains: 1 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array:

 AAGACCAAGG ATCGGGAG 18 SEQ ID NO: 15

Array length: 420

Sequence type: nucleic acid

Number of chains: 2

 Topology: linear

Sequence type: other nucleic acid (PCR amplified DNA fragment)

Array:

 TCTCGTAAAC TCCAGAGCAG CGATAGGCCG TAATATCGGG GAAAGCACTA TAGGGACATG 60 ATGTTCCACA CGTCACATGG GTCGTCCTAT CCGAGCCAGT CGTGCCAAAG GGGCGGTCCC 120 GCTGTGCACA CTGGCGCTCC AGGGAGCTCT GCACTCCGCC CGAAAAGTGC GCTCGGCTCT 180 GCCAGGACGC GGGGCGCGTG ACTATGCGTG GGCTGGAGCA ACCGCCTGCT GGGTGCAAAC 240 CCTTTGCGCC CGGACTCGTC CAACGACTAT AAAGAGGGCA GGCTGTCCTC TAAGCGTCAC 300 CACGACTTCA ACGTCCTGAG TACCTTCTCC TCACTTACTC CGTAGCTCCA GCTTCACCAG 360 ATCTCGGAAT GGACCCCAAC TGCTCCTGCT CCACCGGTAA GACTCCCGAT CCTTGGTCTT 420 sequences N °: 16

Array length: 2160

Sequence type: nucleic acid

Number of chains: 2

Topology: linear Sequence type: genomic DNA

Array:

GGATCCCAAG GCCCAACTCC CCGAACCACT CAGGGTCCTG TGGACAGCTC ACCTAGCTGC 60 AATGGCTACA GGTAAGCGCC CCTAAAATCC CTTTGGCACA ATGTGTCCTG AGGGGAGAGG 120 CAGCGACCTG TAGATGGGAC GGGGGCACTA ACCCTCAGGG TTTGGGGTTC TGAATGTGAG 180 TATCGCCATC TAAGCCCAGT ATTTGGCCAA TCTCAGAAAG CTCCTGGCTC CCTGGAGGAT 240 GGAGAGAGAA AAACAAACAG CTCCTGGAGC AGGGAGAGTG TTGGCCTCTT GCTCTCCGGC 300 TCCCTCTGTT GCCCTCTGGT TTCTCCCCAG GCTCCCGGAC GTCCCTGCTC CTGGCTTTTG 360 GCCTGCTCTG CCTGCCCTGG CTTCAAGAGG GCAGTGCCTT CCCAACCATT CCCTTATCCA 420 GGCTTTTTGA CAACGCTATG CTCCGCGCCC ATCGTCTGCA CCAGCTGGCC TTTGACACCT 480 ACCAGGAGTT TGTAAGCTCT TGGGGAATGG GTGCGCATCA GGGGTGGCAG GAAGGGGTGA 540 CTTTCCCCCG CTGGAAATAA GAGGAGGAGA CTAAGGAGCT CAGGGTTTTT CCCGACCGCG 600 AAAATGCAGG CAGATGAGCA CACGCTGAGC TAGGTTCCCA GAAAAGTAAA ATGGGAGCAG 660 GTCTCAGCTC AGACCTTGGT GGGCGGTCCT TCTCCTAGGA AGAAGCCTAT ATCCCAAAGG 720 AACAGAAGTA TTCATTCCTG CAGAACCCCC AGACCTCCCT CTGTTTCTCA GAGTCTATTC 780 CGACACCCTC CAACAGGGAG GAAACACAAC AGAAATCCGT GAGTGGATGC CTTCTCCCCA 840 GGCGGGGATG GGGGAGACCT GTAGTCAGAG CCCCCGGGCA GCACAGCCAA TGCCCGTCCT 900 TGCCCCTGCA GAACCTAGAG CTGCTCCGCA TCTCCCTGCT GCTCATCCAG TCGTGGCTGG 960 AGCCCGTGCA GTTCCTCAGG AGTGTCTTCG CCAACAGCCT GGTGTACGGC GCCTCTGACA 1020 GCAACGTCTA TGACCTCCTA AAGGACCTAG AGGAAGGCAT CCAAACGCTG ATGGGGGTGA 1080 GGGTGGCGCC AGGGGTCCCC AATCCTGGAG CCCCACTGAC TTTGAGAGAC TGTGTTAGAG 1140 AAACACTGGC TGCCCTCTTT TTAGCAGTCA GGCCCTGACC CAAGAGAACT CACCTTATTC 1200 TTCATTTCCC CTCGTGAATC CTCCAGGCCT TTCTCTACAC TGAAGGGGAG GGAGGAAAAT 1260 GAATGAATGA GAAAGGGAGG GAACAGTACC CAAGCGCTTG GCCTCTCCTT CTCTTCCTTC 1320 ACTTTGCAGA GGCTGGAAGA TGGCAGCCCC CGGACTGGGC AGATCTTCAA GCAGACCTAC 1380 AGCAAGTTCG ACACAAACTC ACACAACGAT GACGCACTAC TCAAGAACTA CGGGCTGCTC 1440 TACTGCTTCA GGAAGGACAT GGACAAGGTC GAGACATTCC TGCGCATCGT GCAGTGCCGC 1500 TCTGTGGAGG GCAGCTGTGG CTTCTAGCTG CCCGGGTGGC ATCCCTGTGA CCCCTCCCCA 1560 GTGCCTCTCC TGGCCCTGGA AGTTGCCACT CCAGTGCCCA CCAGCCTTGT CCTAATAAAA 1620 TTAAGTTGCA TCATTTTGTC TGACTAGGTG TCCTTCTATA ATATTATGGG GTGGAGGGGG 1680 GTGGTATGGA GCAAGGGGCC CAAGTTGGGA AGACAACCTG TAGGGCCTGC GGGGTCTATT 1740 CGGGAACCAA GCTGGAGTGC AGTGGCACAA TCTTGGCTCA CTGCAATCTC CGCCTCCTGG 1800 GTTCAAGCGA TTCTCCTGCC TCAGCCTCCC GAGTTGTTGG GATTCCAGGC ATGCATGACC 1860 AGGCTCAGCT AATTTTTGTT TTTTTGGTAG AGACGGGGTT TCACCATATT GGCCAGGCTG 1920 GTCTCCAACT CCTAATCTCA GGTGATCTAC CCACCTTGGC CTCCCAAATT GCTGGGATTA 1980 CAGGCGTGAA CCACTGCTCC CTTCCCTGTC CTTCTGATTT TAAAATAACT ATACCAGCAG 2040 GAGGACGTCC AGACACAGCA TAGGCTACCT GCCATGGCCC AACCGGTGGG ACATTTGAGT 2100 TGCTTGCTTG GCACTGTCCT CTCATGCGTT GGGTCCACTC AGTAGATGCC TGTTGAATTC 2160 SEQ ID NO: 17

Array length: 19

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array:

 TTCAAGCAGA CCTACAGCA 19 SEQ ID NO: 18

Array length: 17 Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array:

 GAGGTCTGGG GGTTCTG 17 SEQ ID NO: 19

Array length: 18

Sequence type: nucleic acid

Number of chains: 1

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array:

 GGTAGCCGGA TCAAGCGT 18

Claims

The scope of the claims  1. The following steps (A) and (B):  (A) incorporating a gene encoding growth hormone into living stem cells, and  (B) a step of introducing the stem cells obtained in the step (A) into a living body, A growth transformant obtained by the step comprising:  2. The growth transformant according to claim 1, wherein the growth transformant is a mammal, a bird, a reptile, an amphibian or a fish.  3. The growth transformant according to claim 1, wherein the integration of the gene encoding growth hormone into living stem cells is performed using a retrovirus vector.  4. The following steps (A), (B) and (C): '  (A) a step of incorporating a gene encoding growth hormone into living stem cells, (B) a step of introducing the stem cells obtained in the step (A) into a living body, and (C) a step of expressing a growth hormone in a living body obtained in the step (B).  5. The method for producing a growth transformant according to claim 4, wherein the growth transformant is a mammal, a bird, a reptile, an amphibian or a fish.  6. The method for producing a growth transformant according to claim 4, wherein the integration of a gene encoding growth hormone into living stem cells is performed using a retrovirus vector.