Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-06-29
2004-07-06
Reynolds, Deborah J. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S008000, C800S003000
Reexamination Certificate
active
06759568
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-247729, filed Aug. 17, 2000, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to high estrogen-sensitive medaka fish, and more specifically, to transgenic medaka fish having a medaka-derived estrogen receptor gene introduced.
The transgenic medaka fish of the present invention can be used to detect an estrogen-like endocrine disrupting chemicals, and also used as an experimental animal for elucidating development mechanism of thrombosis and as bioassay system for developing a therapeutic agent of thrombosis.
2. Description of the Related Art
Recently, the effects of chemical substances present in the environment upon endocrine system of an organism have been intensively studied, and have attracted a high interest increasingly. Since many chemical substances having endocrine disrupting activity upon an organism exhibit estrogen-like action, most of studies are directed to estrogen which is a female hormone.
Pollution of rivers with such estrogen-like chemical substances has constituted a social problem on a global scale. To detect the estrogen-like chemical substances from a river in the present invention, we tried the use of medaka fish which has excellent features as an experimental animal. However, the medaka fish cannot be used as an aquatic animal for testing environmental water, since it is not sensitive even to an extremely small amount of estrogen. This problem was found for the first time by the present inventor in the course of making the present invention.
BRIEF SUMMARY OF THE INVENTION
The present invention was made to overcome the aforementioned problem. An object of the present invention is to provide transgenic medaka fish having sensitivity to a very small amount of estrogen. Another object of the present invention is to provide a method of producing medaka fish having one or more thrombi by using the transgenic medaka fish, and to provide the medaka fish having one or more thrombi produced by the method. A further object of the present invention is to provide a method of testing an estrogen-like acting substance by using the transgenic medaka fish.
To attain the aforementioned objects, the present inventor have succeeded in preparing transgenic medaka fish having a medaka-derived estrogen receptor gene introduced, namely, high estrogen-sensitive medaka fish.
The present invention was made based upon such achievement.
To be more specific, the present invention was achieved by the means described below.
(1) A polynucleotide having a nucleotide sequence represented by Sequence ID No: 1.
(2) A polynucleotide comprising the nucleotide sequence from 211 to 1935 position represented by Sequence ID No: 1.
(3) A protein having an amino acid sequence encoded by the polynucleotide described in (2).
(4) A recombinant vector containing the polynucleotide described in (1) or (2).
(5) Transgenic medaka fish into which the polynucleotide described in (1) or (2) is introduced.
(6) A method of producing medaka fish having one or more thrombi, comprising the step of raising the transgenic medaka fish described in (5) in the presence of estrogen.
(7) Medaka fish having one or more thrombi, which is obtained by raising the transgenic medaka fish described in (5) in the presence of estrogen.
(8) A method of testing an estrogen-like action in test water, comprising the steps of:
raising the transgenic medaka fish described in (5) in the test water; and
observing whether or not one or more thrombi are formed in the medaka fish after the raising step.
(9) The method described in (8), wherein the test water is water taken from environment.
(10) The method described in (8), wherein the test water is water having a test substance added.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
DETAILED DESCRIPTION OF THE INVENTION
Now, the present invention will be explained in detail.
In the present invention, the medaka fish to be used in cloning an estrogen receptor gene and the medaka fish to be used in introducing the cloned gene are not particularly limited, as long as they belong to a species
Oryzias latipes
. The medaka fish actually used in the present invention were obtained from the BioScience Center, Nagoya University. The obtained medaka fish were grown while feeding Tetramin (Tetra) in an amount of about 1-10 mg/day.
It should be noted that fertilized eggs of the medaka fish cannot be deposited, because a technology for resuming the development of a freeze-stored egg, as the need arises, has not yet been established. Therefore, the medaka fish used in the present invention are now raised under control of the present inventor with responsibility. As described in the above, the medaka fish to be used in the present invention are not limited to those raised by the present inventor, and any medaka fish may be used in the present invention.
{Cloning of Medaka-Derived Estrogen Receptor Gene}
The medaka-derived estrogen receptor gene of the present invention is cloned from liver cDNA library of adult medaka fish. More specifically, the medaka-derived estrogen receptor cDNA is cloned by preparing a probe based on a nucleotide sequence of a human-derived estrogen receptor gene and screening the above cDNA library by use of the probe.
The nucleotide sequence of the cloned estrogen receptor cDNA is determined, and the amino acid sequence predicted from the nucleotide sequence is determined. The nucleotide sequence of the medaka-derived estrogen receptor cDNA and the amino acid sequence are shown by Sequence ID No: 1 and Sequence ID No: 2, respectively in the Sequence Listing.
In the present invention, the nucleotide sequence for expressing medaka estrogen receptor may have arbitrary length, as long as it comprises at least a coding region (i.e., the nucleotide sequence from 211 to 1935 position represented by Sequence ID No: 1). Furthermore, in the present invention, the amino acid sequence of the medaka estrogen receptor may also have deletion, addition and/or substitution of one or several amino acids in the amino acid sequence represented by Sequence ID No: 2, as long as it has the same function as the protein consisting of the amino acid sequence represented by Sequence ID No: 2.
{Preparation of Recombinant Vector}
The medaka-derived estrogen receptor cDNA cloned by the aforementioned method is introduced into a vector. The introduction of the cDNA into a vector can be performed in accordance with a known genetic engineering process. In this manner, it is possible to prepare a recombinant vector into which the medaka-derived estrogen receptor gene is inserted.
The vector to be used in the present invention is not particularly limited, as long as it can express the protein encoded by a foreign gene inserted therein. In the present invention, it is preferable to use a plasmid having a promoter sequence and a poly (A) signal sequence, as a vector. For example, as described later in examples, a new plasmid vector is constructed by purifying each DNA fragment from another plasmid containing a DNA fragment of a medaka actin promoter and from another plasmid containing a DNA fragment of a SV40 poly (A) signal, and the resultant plasmid vector may be used.
{Preparation of Transgenic Medaka Fish}
The recombinant vector prepared as mentioned above is transferred into a nucleus of a medaka fertilized egg, thereby preparing transgenic medaka fish capable of expressing the estrogen receptor gene in an excessive amount. As the medaka fertilized egg to be transformed in the present invention, an embryo at a sin
President of Hiroshima University
Reynolds Deborah J.
Woitach Joseph
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