Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-07
2001-03-27
Clark, Deborah J. R. (Department: 1633)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S455000, C800S025000, C800S003000, C536S024100
Reexamination Certificate
active
06207817
ABSTRACT:
FIELD OF THE INVENTION
This invention (published in
DNA and Cell Biology
(1998), 17:359-376, and
Bioindustry
(1998), 9:1-9, hereinafter incorporated by reference) relates to the findings of the DNA sequences of fish insulin-like growth factor II (IGF-II) promoter regions and recombinant IGF-II promoters and the expression of said IGF-II promoter regions and recombinant IGF-II promoters in eukaryotic cells and fish embryos of another fish species. The integration of said IGF-II promoter regions and recombinant IGF-II promoters into the somatic and germ cells of fish from another species results in the creation of a transgenic fish. The results of this invention demonstrate that fish IGF-II promoter regions and recombinant IGF-II promoters not only are acting as a growth factor which can stimulate the growth and development of fish, but also are capable of being expressed in eukaryotic cells other than fish, such as in human lung large cell carcinoma cells.
BACKGROUND OF THE INVENTION
Insulin-like growth factors (IGFs) are mitogenic peptide hormones. There are two kinds of IGFs, namely, IGF-I and IGF-II. These two polypeptides have high homology of protein folding structure and play important regulatory roles in growth and differentiation of vertebrates.
The gene structure of IGF-I has been reported in mammals (e.g., humans, rats, sheep) and fish (e.g., salmon). The mature form of the IGF-1 peptide is a 70 amino acid polypeptide which mediates the growth-promoting actions of growth hormone as well as having important local paracrine and autocrine roles in multiple organs (Kavsan et al. (1993),
DNA and Cell Biology,
12:729-737).
The gene structure of IGF-II has been analyzed in various mammals including humans. Until recently, there has been no report relating to the gene structure of IGF-II in fish. The inventors of the present invention are the first to have discovered the IGF-II gene structure in fish (Chen et al.,
DNA and Cell Biology
(1997), 16:883-892). Their findings demonstrate that the gene structure of fish IGF-II is different from that in mammals. For example, human IGF-II gene consists of 10 exons about 30 kb in length, which encode a mature, circulating polypeptide approximately 70 amino acids in length (Gray et al. (1987),
DNA,
6:283-295). In contrast, the mature form of fish IGF-II polypeptide is contained in 4 exons about 13 kb in length, although it is also approximately 70 amino acids in length (Chen et al.,
DNA and Cell Biology
(1997), supra).
The mature form of IGF-II polypeptide in different fish species is highly conserved, suggesting that an IGF-II polypeptide derived from one fish species may display similar growth promotion effects on another fish species.
Although IGF-I and IGF-II share high homology of protein folding structure and similar growth promotion effects, their biologic effects are mediated by different IGF receptors. The IGF-I receptor is a tyrosine kinase receptor; the IGF-II receptor is a mannose-6-phosphate receptor.
Recently, studies regarding the findings of fish IGF-I promoters have been reported by several groups of investigators (Koval et al. (1994),
DNA and Cell Biology,
13:1057-1062; Kulik et al. (1995),
J. Biol. Chem.,
270:1068-1073). These reports show that the IGF-I promoters have potent stimulatory activity in cells, suggesting that the IGF-I promoters may play a regulatory role in stimulating the expression of IGF-I in cells or tissues by providing cells or tissues with transcription factor binding, especially of the liver-specific transcription factor.
So far, no study, other than the one to be presented by the inventors in the present invention, has been reported concerning the IGF-II promoter region(s) in fish.
In the invention to be presented below, the inventors will describe their findings of the DNA sequences of the IGF-II promoters in fish. Because these inventors also have completed substantial research on the IGF-I promoters and because IGF-I and IGF-II have been regarded as possessing similar regulatory functions, the inventors will present their findings of IGF-I promoters together with those of IGF-II promoters in the following sections, particularly for comparison purpose. These inventors will show that fish IGF-II promoters not only affect the growth of fish embryos earlier than IGF-I promoters but also display higher levels of gene expression than IGF-I promoters. The inventors will also show that an IGF-II promoter from one fish species not only can be expressed in eukaryotic cells other than fish, such as human cells, but also can be integrated into the somatic and germ cells of another fish species, thus creating a transgenic fish.
SUMMARY OF THE INVENTION
The present invention includes the following findings: (1) identification and characterization of the promoter regions of fish IGF-II gene (there are two IGF-II promoter regions upstream from the IGF-II gene, namely, the first and the second IGF-II promoter regions); (2) determination of the promoter activity in different segment or segments of the IGF-II promoter regions (hereinafter referred to as “the recombinant IGF-II promoters”) by inserting said recombinant IGF-II promoter segment(s) into a vector to form a plasmid construct and transfecting said plasmid construct into eukaryotic cell lines; (3) expression of fish IGF-II promoter regions or recombinant IGF-II promoters in eukaryotic cell lines; and (4) expression of fish IGF-II promoter regions or recombinant IGF-II promoters in fish embryos of another species, thus, creating a transgenic fish.
There are two promoter regions in IGF-II gene which have demonstrated promoter activities, namely, the first and the second IGF-II promoter regions. The first promoter region has the DNA sequence of SEQ ID NO:1, and the second promoter region has the DNA sequence of SEQ ID NO:13.
Relatively high promoter activities have been found in some segments of the two IGF-II promoter regions. For instance, the first IGF-II promoter region comprises several segments which, individually or in combination of several segments, have shown promoter activities. These segments, which collectively called “the recombinant IGF-II promoters”, have the DNA sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The preferable recombinant IGF-II promoters (i.e., showing high promoter activities) include a segment having DNA sequence of SEQ ID NO:6 and segments containing DNA sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
In order to detect the promoter activity and mass produce IGF-II promoter region and recombinant IGF-II promoters, 6 synthetic primers are designed. These 6 primers have the DNA sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
To test the promoter activity of either a fish IGF-II promoter region and a recombinant IGF-II promoter in eukaryotic cells, the IGF-II promoter region or recombinant IGF-II promoter is first amplified by polymerase chain reaction (PCR) method using the above mentioned 6 synthetic IGF-II primers; the amplified promoter sequence is ligated with a chloramphenicol acetyltransferase (CAT) coding sequence, and then inserted into a vector to form a plasmid construct; the plasmid construct is transfected into human lung large cell carcinoma cells. The relative promoter activity is measured by CAT assay.
The expression of a fish IGF-II promoter region or a recombinant IGF-II promoter in eukaryotic cells is carried out by ligating the IGF-II promoter region and recombinant IGF-II promoter with an IGFs promoter-driven green fluorescent protein (GFP) and a vector to form a plasmid construct. Said plasmid construct is then transfected an eukaryotic cells. The promoter expression is determined by monitoring the fluorescence under a fluorescence microscope or by RT (reverse transcription)—PCR. At least three kinds of eukaryotic cells, including tilapia ovary TO-2 cells, chinook salmon embryo CHSE-214 cells, and human lung large cell carcinoma cells are used for this study.
The expression of a fish IGF-II prom
Chen Jyh-Yih
Wu Jen-Leih
Academia Sinica
Chao Fei-Fei
Chen Shin-Lin
Clark Deborah J. R.
Venable, Baetjer, Howard & Civiletti
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