Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-11-14
2003-10-21
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S024100
Reexamination Certificate
active
06635474
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a mammary gland tissue-specific expression system using the promotor site for the &bgr;-casein gene of Korean native goats, through which physiological activating substances can be produced. More particularly, the present invention relates to novel recombinant mammalian expression vectors in which a &bgr;-casein gene expression-regulating region, a physiological activating substance gene and a termination-regulating region are linked. Also, the present invention is concerned with a method for producing physiological activating substances in mammary gland tissue-derived cell lines and in animals, using the novel recombinant vectors.
BACKGROUND ART
Physiological activating substances are produced and secreted at trace amounts in the human body and play an essential role in various metabolisms and modulations. The physiological activating substances known to date include insulin, interleukins, hemopoietic growth regulating factors, such as stem cell factor, granulocyte colony stimulating factor, erythropoietin, etc, and are too numerous to describe their great functions in the human body, in detail. The reason why such physiological activating substances, in spite of their importance, have not yet been industrialized, is that they are difficult to isolate and purify owing to their trace amount in the human body. Further, the physiological activating substances produced by using a procaryotic expression system, such as that obtained from
E.coli
, frequently do not perform their normal functions in the human body as well as have not yet overcome the safety problem which must be solved before administration.
According to the reports, contributed to the academic circles, it is known that, even if a promoter site is used site for a gene which is expressed specifically in a mammary gland tissue, the expression level is different depending on the species from which the promotor is obtained and on the genes to be expressed (Clark et al. (1987)
Trends Biotech.
5, 20-24; Simons et al., (1987)
Nature
328, 530-532; Lee at al., (1988)
Nucl. Acids Res.
16, 1027-1041; Vilotte et al., (1988)
Eur. J. Biochem.
186, 43-48; Gorden et al., (1987)
Bio/Technology
8, 443-446; Shani et al., (1992)
Tragenic Res.
1, 195-208; Wright el al., (1991)
Bio/Technology
9, 830-834; Ebert et al., (1991)
Bio/Technology
9, 835-838; Mega et al., (1994)
Transgenic Res.
3, 36-42Wei et al., (1995)
Transgenic Res.
4, 232-240; Gutierrez et al., (1996)
Transgenic Res.
5 271-279)
In order to produce physiological activating substances, the expression systems which take advantage of
E. coli
(Korean Pat. Publication No. 94-5585) and animal cells have been usually used. These techniques would occasionally bring about industrial successes, but still have significant problems to be solved. For instance, in the case of the expression utilizing
E. coli
, mass production is possible with low cost. However, since
E. coli
, a prokaryote, does not perform a posttranslational modification, which is a feature of eukaryotes, such a human physiological activating substance as EPO cannot exert its activity if it is produced in
E.coli
. To avoid this problem, active research has been and continues to be directed to the development expression systems which take advantage of animal cells. The products expressed in these systems are active in the human body because they experience posttranslational modifications. However, the problem of high cost for culturing animal cells remains unsolved.
Almost all of the physiological activating substances that are industrially produced utilize the above mentioned techniques. so they have the problems to be solved, including activity sustenance, cost, and isolation and purification.
DISCLOSURE OF THE INVENTION
The mammary gland-specific expression systems developed by the present inventors, named pGbc, pGbc_L and pGbc_S were deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure in the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology at 52, Oun-dong, Yusong-Ku, Taejon 305-333, Republic of Korea, on Aug. 17, 1998, and the accession (deposit) Nos. KCTC 0515BP, 0514BP AND 0513BP were issued, respectively. All restrictions on the availability to the public of the deposited materials will be irrevocably removed upon the granting of a patent. The systems of the present invention make it possible that desired proteins are produced by expression in mammary gland tissue-derived animal cells or through the milk secreted from the transgenic animals with the expression systems, thereby solving the above-mentioned problems, that is, the activity sustenance, production cost, and isolation and purification of the desired proteins.
Use of the expression-regulating region of a &bgr;-casein gene, expressed specifically in mammary gland tissues, in producing human physiological activating substances, brings about the following industrial advantages. First, because the target proteins which arc produced by the recombination technique of the present invention experience the same postranslational modification as that which the corresponding naturally occurring proteins do, the target proteins can sustain their activity in the human body. Secondly, by virtue of taking advantage of specificity for mammary gland tissue, the expression systems of the present invention employing mammary gland tissue-derived cells or transgenic animals can produce physiological activating substances at much lower cost than do the expression systems using general animal cells. The proteins produced in mammary gland tissue-derived cells or through the milk secreted from transgenic animals are few in number, so that the target protein is easy to isolate and purify. Additionally, transgenic animals require no further significant cost in scaling-up the production of the target proteins as well as produce no pollution during its production. A third advantage is the safety of the physiological activating substances produced. Because there are no toxins in the products secreted from the mammary gland tissues, the expression system of the present invention is safer than other conventional systems.
In order to producing physiological activating substances, methods utilizing
E.coli
, or animal cells were developed and more recently, advantage has been taken of transgenic animals. The expression techniques using
E.coli
as a host or using animal cells have now a limitation in industrial application owing to the above-mentioned problems, that is, the activity sustenance, production cost, and isolation and purification of the physiological activating substances produced. As a measure of settling these problems, transgenic animal and related techniques have rapidly developed and now make a great advance in biological studies.
The present invention uses a mammary gland tissue-derived cell line and a transgenic animal in producing proteins. For this, molecular biological technology and other apical techniques are employed in the present invention. For example, DNA recombination techniques are needed for constructing the mammalian expression vectors which are able to be expressed specifically in mammary gland tissues and a microinjection technique is for producing a transgenic animal with the vectors.
Although it is well known to those skilled in the art to use the promoter sites of the genes expressed specifically in mammary gland tissues in constructing a mammalian expression vector which is able to express proteins specifically in the mammary gland tissues and the mammalian expression vector of the present invention is originated from pRC/RSV, a commercial vector (Invitrogen Inc.), the expression systems of the present invention is quite different from those of other conventional techniques in the following aspects. First, the goat &bgr;-casein promoter used in the present invention is obtained from Korean native goats. A second characteristically different
Han Young Mahn
Jeong Hae Young
Kim Sun Jung
Ko Jung Ho
Lee Kyung Kwang
Falk Anne-Marie
Hanmi Pharm Co., Ltd
Knobbe Martens Olson & Bear LLP
Qian Celine
LandOfFree
Mammary gland tissue-specific expression system using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Mammary gland tissue-specific expression system using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mammary gland tissue-specific expression system using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3164530