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Transgenic avian which has foreign gene containing sequence encoding feline-derived protein and method for production thereof

a technology a production method, which is applied in the field of transgenic birds, can solve the problems of difficult to say that what was possible with a human-derived protein is also possible, and difficulty in obtaining a high level of expression of a foreign gene in animal cells,

Inactive Publication Date: 2009-06-18
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0096]According to the present invention, transgenic birds capable of producing a feline-derived protein and a method of production thereof are provided. As a result, transgenic birds producing a feline-derived cytokine and a method of production thereof are provided. Furthermore, transgenic birds producing a feline-derived erythropoietin and a method of production thereof are provided. Transgenic birds producing erythropoietin at higher concentrations than in the prior art and the production thereof have become possible.BEST MODES FOR CARRYING OUT THE INVENTION
[0097]The following examples illustrate the present invention in detail. These examples are, however, by no means limitative of the scope of the present invention. Unless otherwise specifically described, gene manipulation procedures were carried out according to the typical methods (J. Sambrook, E. F. Fritsch, T. Maniatis; Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory). Unless otherwise specifically described, cell cultures were carried out according to the typical methods (KOYAMA, Hideki (ed.): “Saibo-Baiyo Labo Manual (Cell Culture Labo Manual)”, Springer Verlag Tokyo, 1st Ed.). When trademarks are given, the instructions given in the manuals attached were followed, unless otherwise specifically described.

Problems solved by technology

It is therefore very difficult to obtain a high level of expression of a foreign gene in animal cells.
Further, since there is a difference in sugar chain sequence between human and cat, it is difficult to say that what was possible with a human-derived protein is also possible with the corresponding feline-derived protein.
The human-derived erythropoietin-producing transgenic birds disclosed in United States Patent Application Publications 2004 / 0019922 and 2004 / 0019923 have a problem in that the erythropoietin production is low.

Method used

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  • Transgenic avian which has foreign gene containing sequence encoding feline-derived protein and method for production thereof
  • Transgenic avian which has foreign gene containing sequence encoding feline-derived protein and method for production thereof
  • Transgenic avian which has foreign gene containing sequence encoding feline-derived protein and method for production thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of feline-derived erythropoietin gene expression plasmid pMSCVneobactfEPO

[0098]pMSCVneobact (SEQ ID NO:2) was totally synthesized based on the relevant prior art document (Gene Ther. 1994, Mar.: 1(2):136-8) and internet information (http: / / www.ncbi.nlm.NIH.gov / etc.) and inserted into pUC19 (GenBank Accession No. X02514) at a site between EheI (235) and PvuII (628) (products of Toyobo). The product was cleaved with HindIII (product of Takara Bio) and, after treatment with Alkaline Phosphatase BAP (product of Takara Bio), the desired fragment was purified and recovered using MinElute Reaction Cleanup Kit (product of QIAGEN). This was electrophoresed on 1% agarose and the desired fragment was purified and recovered using MinElute Gel Extraction Kit (product of QIAGEN) (Example 1 vector fragment).

[0099]pUCfEPO (SEQ ID NO:3) encodes, at 911 to 1489 bp, the feline-derived erythropoietin sequence. A fragment amplified by PCR using Pyrobest DNA polymerase (product of Takara Bi...

example 2

Retrovirus Vector Preparation Using pMSCVneobactfEPO and pVSV-G

[0102]Hereinafter, unless otherwise specified, the medium used was Dulbecco's Modified Eagle Medium (DMEM) (product of Gibco) containing 10% of fetal bovine serum (FBS) and 50 units / ml each of penicillin and streptomycin. The cultivation was carried out at 37° C. in the presence of 5% CO2. The plasmid DNA used in the retrovirus vector was Endo Free Plasmid Maxi Kit (product of QIAGEN).

[0103]For retrovirus vector preparation from the plasmid pMSCVneobactfEPO constructed in Example 1, a collagen-coated culture dish having a diameter of 100 mm was sowed with GP293 packaging cells having the gag and pol genes (5×106 cells / dish; 70% confluent) (90% confluent on the next day). On the next day, the medium was removed, and 7.2 ml of the medium and 10 μl of 25 mM chloroquine (product of Sigma) were added, followed by further 1 hour of cultivation. A 56-μl portion of Lipofectamine 2000 (product of Invitrogen) was suspended in 1.4 ...

example 3

Virus Titer Measurement

[0105]The virus titer is defined as the number of infected cells after addition of the virus-containing fluid to NIH3T3 cells (American Type Culture Collection CRL-1658). A 1-ml portion of the virus solution of Example 2 as diluted at a dilution ratio of 102 to 106 was added to 5×104 NIH3T3 cells contained in each well (base area about 9.4 cm2) of each 6-well culture plate, and the proportion of cells expressing the neomycin resistance gene as a marker was determined based on the resistance to G418. If 4 colonies appear at a dilution ratio of 106, the virus titer will be 4×106 cfu / ml.

[0106]More specifically, 6-well culture plates were sowed with 5×104 NIH3T3 cells per well on the day before the start of titer measurement, and the cells were cultured. On the next day, the cell culture medium was replaced with 900 μl of the medium containing 9 μg / ml of polybrene, the virus-containing fluid was diluted to 10−1 to 10−5 with the medium and 100-μl portions of each d...

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Abstract

The present invention has for its object to provide a transgenic bird with a foreign gene containing a feline-derived protein-encoding sequence as transferred therein, and a method of producing the same. The present invention provides a method of producing a feline-derived protein by using a transgenic bird with a method which comprises infecting an avian embryo with a replication defective retrovirus vector containing a foreign gene by microinjection thereof into the early heart or blood vessel formed in the embryo and allowing the embryo to hatch.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of producing a transgenic bird containing a foreign gene as transferred into the genome thereof and to the expression of a feline-derived protein in such transgenic bird. More particularly, it relates to the expression of feline-derived erythropoietin in such transgenic bird.BACKGROUND ART[0002]In recent years, a number of proteins have come into use as pharmaceuticals. This is because the gene recombination technology has been developed for and applied to the introduction or transfer of a gene coding for a desired protein into microorganisms or mammalian cells, so that commercial protein production is now feasible by cultivating the thus-produced genetically modified organisms. For such a medicinal protein to show the physiological activity or activities intrinsic therein, posttranscriptional modifications, for example folding, glycosylation and disulfide bond formation is necessary as in nature.[0003]Methods of produci...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C07K14/00
CPCA01K67/0275A01K2217/05A01K2227/30A01K2267/01C12N2799/027A61K38/00A61K47/48215C07K14/505C12N15/8509A01K2267/02A61K38/1816A61K47/60A61P7/06A01K2217/052C07K14/005C12N7/00C12N15/86C12N2015/8518C12N2740/10043C12N2740/10052C12N2760/20222C12N2830/008
Inventor NAKAISHI, TOMOYUKISHINDO, TAKUYAAWA, TOMOKO
Owner KANEKA CORP
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