Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-11-19
2001-05-08
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000
Reexamination Certificate
active
06229070
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method of gene transfer, which enables stable expression of an exogenous gene in a plant cell.
2. Description of Related Art
Many kinds of transgenic plants with an exogenous gene introduced have been produced to render various characteristic to a plant. In the production of transgenic plants, the diversity of gene expression is observed among individual transgenic plants. It is considered that the diversity is caused by the position of introduced gene in a chromosome. When an exogenous gene is introduced into active chromatin region, high expression of the exogenous gene would be obtained. On the contrary, when an exogenous gene is introduced into inert chromatin region, sufficient expression of the gene would not be obtained (Galli, Current opinion in plant technology (1998) 1:166-172, Matzke et al., Current opinion in plant technology (1998) 1:142-148). Such effect described above is called “position effect”. Because of the position effect, a transgene, which is introduced into a plant, exhibits absolute failure of expression, only weak expression or suspension of expression caused by plant growth or exogenous environment. This phenomenon serves as a barrier to commercialization of a transgenic plant and establishment of a method to stabilize expression of an introduced gene have been demanded.
Recently, some transformants, wherein genes of various kinds are introduced, revealed constant expression of the gene independent of its introduced position in the chromosome. The factors concerning such phenomenon are classified into three cases. These are, insulator, LCR (locus control region) and MAR (matrix attachment region) and involvement of such factors on constant expression is suggested. These three factors function as boundaries in a chromosome and blockade effects of near-existing chromatin, though the mechanism of action of these factors differs from each other.
When an insulator is located between an enhancer and a promoter, the insulator operates to blockade the effect of the enhancer as a silencer. In a higher eukaryotic plant, an enhancer and a silencer might cause an effect on transcriptional activity of not only a certain promoter, but also plural promoters. Therefore, plural elements might cause effects randomly in a chromosome. This phenomenon might work to render diversity on gene expression. On the other hand, a mechanism to restrict the enhancer function or the silencer function might be necessary for precise regulation of gene expression. At present, it is speculated that, an insulator might operate to restrict these functions.
LCR is a region higher-sensitive to DNasel. LCR might form active chromatin, which facilitates physical accession of promoter and various transcriptional factors.
MAR contains an adenine, thymine (AT) rich sequence and a topoisomerase II recognition sequence. Moreover, MAR exhibits nuclear-membrane binding activity under in virto condition. MAR is considered to exist more than every 10-100 kb in a chromosome, and the chromosome binds to nuclear membrane through these regions to form conformation of the chromosome. MAR is indispensable for compaction of chromosome in a limited space of nucleus. The knowledge indicating that the MAR might be involved in the regulation of gene expression is accumulating in recent researches. For example, the analysis of MAR derived from kappa light chain immunogloblin gene of mouse revealed following knowledge.
(1) MAR is necessary for gene expression in the process of development.
(2) Inactivation of gene expression is caused by deletion of MAR through high-order methylation of DNA.
Moreover, a transcriptional unit of one or more gene is contained in a chromatin roop formed among plural MAR's. The analysis of beta globin gene wherein a cluster is formed in the loop revealed that, mutual affinity of each gene was indispensable for appropriate regulation and function of the gene. From these knowledge, it is speculated that, one chromatin loop among MAR's might form an independent unit, and gene expression might be regulated per the unit.
As described above, stable expression of introduced gene is demanded to obtain a practical transformant, regardless of whether the transformant is an animal or a plant. As described heretofore, avoidance of effect caused by surroundings of introduced location, a position effect, is indispensable. Therefore, the possibility that the position effect might be avoided by insertion of LCR, MAR or insulator have been investigated. In animal cell, LCR and insulator functioned as expected and the diversity of gene expression among transformants decreased (Akasaka, Cell Engineering (1997)16:1476-1484, Yasue et al. JP 6550/97). Concerning MAR, investigations performed by a promoter gene or a reporter gene did not exhibit consistent results. Therefore, stable expression of introduced gene was not achieved in many cases. In plants, this result might be caused by following phenomenon.
(1) Plenty of MAR's might operate to increase expression of introduced gene.
(2) When plural copies are introduced, expression of introduced gene might be suppressed by other effects than position effect, for example, specific methylation of DNA.
Therefore, MAR might not function as a boundary on the chromosome necessarily (Galli, Current opinion in plant technology (1998)1:166-172, Matzke et al., Current opinion in plant technology (1998)1:142-148), resulting in failure of generalization of the technique using MAR.
SUMMARY OF THE INVENTION
Therefore, the inventors paid attention to an insulator, which blockades the function of an enhancer and a silencer. There is no prior investigation concerning the effect of an insulator on the expression of a transgene in a plant. Then availability of insulator in a plant remained to be unknown. In this invention, an insulator was adopted to decrease the position effect of a transgene in a plant. Expression of an introduced gene was stabilized in a cultured tobacco cell, without increase of the expression of the gene. This invention provides a method for stable expression of a transgene in a plant.
Hitherto, insulators have been identified from various organisms, for example, Drosophila. The examples of insulators identified are, gypsy insulator, scs-scs' insulator and Fab-7 insulator originated from Drosophila, beta globin insulator originated from chicken, apoB insulator originated from chicken and human. The identification of proteins involved in insulator function have been performed recently, in particular, the analysis on gypsy insulator, scs-scs' insulator is advanced.
This invention provides a method for stable expression of an exogenous gene in a plant cell or in a plant body. It was achieved by concurrent introduction of an exogenous gene and an insulator derived from an organism of heterogeneous species against said plant. Moreover, this invention provides a transgenic plant comprising a plant having an exogenous gene and an insulator. Specifically, the insulator derived from sea urchin arylsulfatase gene was adopted in this invention.
Theoretically, any plant can be adopted as a host plant to be introduced an exogenous gene. The examples of plants preferred to be adopted as a host plant are as described below. These are useful cultivated plants such as tobacco, Arabidopses or petunia, crops such as rice, maize, potato, sweet potato, soybean, strawberry or eggplant and trees such as blue gum or white poplar.
Moreover, any gene can be adopted as an exogenous gene to be introduced in a host plant. The examples of exogenous genes preferred to be introduced in a host plant are as described below. These are disease or insect injury resistance genes such as peroxidase gene or chitinase gene, genes for ectoine biosynthesis such as L-2,4-diaminobutyric acid acetyltransgerase, L-2,4-diaminobutyric acid transaminase and ectoine synthetase, genes for betaine biosynthesis such as choline oxidase and second metabolite producing gene such as fatty acid biosynthesis
Akasaka Koji
Kato Ko
Kusumi Takaaki
Shinmyo Atsuhiko
Tanaka Yoshikazu
Burns Doane Swecker & Mathis L.L.P.
Kruse David
Nara Institute of Science and Technology
Nelson Amy J.
LandOfFree
Method of stable gene expression in a transgenic plant... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of stable gene expression in a transgenic plant..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of stable gene expression in a transgenic plant... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2538070