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Genetic Insulator for Preventing Influence by Another Gene Promoter Susheng Gan University of Kentucky

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Genetic Insulator for Preventing Influence by Another Gene Promoter Susheng Gan University of Kentucky University of Kentucky UKnowledge Plant and Soil Sciences Faculty Patents Plant and Soil Sciences 10-20-2009 Genetic Insulator for Preventing Influence by Another Gene Promoter Susheng Gan University of Kentucky Mingtang Xie Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Follow this and additional works at: https://uknowledge.uky.edu/pss_patents Part of the Plant Sciences Commons Recommended Citation Gan, Susheng and Xie, Mingtang, "Genetic Insulator for Preventing Influence by Another Gene Promoter" (2009). Plant and Soil Sciences Faculty Patents. 35. https://uknowledge.uky.edu/pss_patents/35 This Patent is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Plant and Soil Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. US007605300B2 (12) United States Patent (10) Patent N0.: US 7,605,300 B2 Gan et al. (45) Date of Patent: Oct. 20, 2009 (54) GENETIC INSULATOR FOR PREVENTING Gan, S. Ph.D. Thesis; Molecular characterization and genetic INFLUENCE BY ANOTHER GENE manipulation of plant senescence (University of Wisconsin-Madi PROMOTER son, Madison, 1995).* Millar et al., The Plant Cell, vol. 11, pp. 825-838, May 1999. (75) Inventors: Susheng Gan, Lexington, KY (U S); NCBI, GenBank, AF129511, 2000. An G, “Binary Ti vectors for plant transformation and promoter Mingtang Xie, Burnaby (CA) analysis.” In Methods in Enzymology.‘ RecombinantDNA, R. Wu, and L. Grossman, eds, 292-305 (San Diego, Academic Press, 1987). (73) Assignee: University of Kentucky Research Bechtold N et al., “In Planta Agrobacterium-mediated gene transfer Foundation, Lexington, KY (U S) by in?ltration of adult Arabidopsis plants”, C R Acad Sci Paris 316, 1194-1199 (1993). ( * ) Notice: Subject to any disclaimer, the term of this Brown M et al., “lac repressor can regulate expression from a hybrid patent is extended or adjusted under 35 SV40 early promoter containing a lac operator in animal cells”, Cell USC 154(b) by 218 days. 49, 603-612 (1987). Gallic DR et al., “The 5‘-leader sequence of tobacco mosaic virus (21) Appl.No.: 11/328,226 RNA enhances the expression of foreign gene transcripts in vitro and in vivo”. Nucleic Acids Res 15, 3257-3273 (1987). (22) Filed: Jan. 10, 2006 Gan S. Ph.D. Thesis; Molecular characterization and genetic manipulation of plant senescence (University of Wisconsin-Madi (65) Prior Publication Data son, Madison, 1995). Hajdukiewicz P et al., “The small, versatile pPZP family of US 2006/0174370 A1 Aug. 3, 2006 Agrobacterium binary vectors for plant transformation”, Plant Mol Biol 25, 989-994 (1994). Related US. Application Data Holtorf S et al., “Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis (62) Division of application No. 09/973,945, ?led on Oct. thaliana Plant Mol Biol 29, 637-646 (1995). 11, 2001, noW abandoned. Jefferson RA, “Assaying chimeric genes in plants: the GUS gene (60) Provisional application No. 60/241,735, ?led on Oct. fusion system”, Plant Mol Biol Rep 5, 387-405 (1987). 20, 2000. Sambrook J et al., Molecular Cloning.'A LaboratoryManual, Second edition (New York, Cold Spring Harbor Laboratory Press, 1989). (51) Int. Cl. Slomiany BA, “Extraction of nuclear proteins With increased DNA binding activity”, BioTechniques 28, 938-942 (2000). C12N 15/82 (2006.01) Mlynarova et a1. Reduced Position Effect in Mature Transgenic (52) US. Cl. .................................... .. 800/278 Plants Conferred by the Chicken LysoZyme Matrix-Associated (58) Field of Classi?cation Search ..................... .. None Region. The Plant Cell. Mar. 1994. vol. 6, pp. 417-426, see entire See application ?le for complete search history. document. (56) References Cited * cited by examiner U.S. PATENT DOCUMENTS Primary ExamineriDavid H Kruse 5,610,053 A 3/1997 Chung et a1. (74) Attorney, Agent, or FirmiMcDermott Will & Emery 6,037,525 A 3/2000 Thompson et a1. LLP 6,040,185 A 3/2000 Dietrich et a1. 6,100,448 A 8/2000 Thompson et a1. (57) ABSTRACT 6,229,070 B1 5/2001 Shinmyo et a1. 6,388,170 B1* 5/2002 Gan et a1. ................. .. 800/278 A 16 bp polynucleotide sequence ofArabidopsis thaliana is a FOREIGN PATENT DOCUMENTS genetic insulator that can effectively isolate a trans gene from positional effects of neighboring gene activities in transgenic W0 WO 93/02187 2/1993 plant cells. OTHER PUBLICATIONS Millar et a1. (1999, The Plant Cell 11:825-838).* 4 Claims, 4 Drawing Sheets US. Patent 0a. 20, 2009 Sheet 1 of4 US 7,605,300 B2 Figure 1 4§_2\P 3158 _ 51 __> H MAS-terl Gus “l'l P358 NPTII 1») FB 44/ LB Insert 1- pGL429 2. pGL450 IE] 3. pGL419 2x Insulator P 4. pGL469 [24x Insulator sis-5. » }—'l. (is | MAS-ter H P353 NPTII 1+ 9 LB Insert 5- pGL443 2x Insulator 6. pGL446 US. Patent 0a. 20, 2009 Sheet 2 of4 US 7,605,300 B2 959“.m US. Patent 0a. 20, 2009 Sheet 3 of4 US 7,605,300 B2 Figure 3 A Nl29 L31 -++++++-NPS e3 CD 0 %ofCXinLan mA o O 12345678 US. Patent 0a. 20, 2009 Sheet 4 of4 US 7,605,300 B2 Figure4 A M29 5 ' cttctaGAATATATATATATTCgaaaaag3 ' Nlm 5- ------------ ——G ------------ --3' M1 5' ---- --Acc ------------------ --3' M2 5' ------- -—GCG --------------- --3' M3 5' ---------- -—CGCG ----------- "3' M4 5' ---------- --c -------------- --3 M5 5' ----------- ——G ------------- --3' M6 5' ------------ --c ------------ --3 M7 5' ------------- —-G ----------- --3 D E’ +100- 200 g 2 80- 150 E . X 60 100 l 0 1234567891012345678910 US 7,605,300 B2 1 2 GENETIC INSULATOR FOR PREVENTING having the sequence set forth in SEQ ID NO:9 INFLUENCE BY ANOTHER GENE (5'GAATATATATATATTC3') or a polynucleotide region hav PROMOTER ing a sequence that is a variant or fragment of the sequence set forth in SEQ ID N019, Wherein the polynucleotide region has RELATED APPLICATIONS a genetic insulator activity. In various embodiments, the poly nucleotide has a sequence as set forth in SEQ ID NOS: 1, 5, 9, This application is a divisional of application Ser. No. 10, 11, 12, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 09/973,945, ?led Oct. 11, 2001 noW abadoned, Which claims 33, 34, 35 or 36. In one embodiment, the plant genetic insu priority of US. Application No. 60/241,735, ?led Oct. 20, lator sequence contains only 16 base pair (bp), and is com 2000, the contents of Which are hereby incorporated by ref pletely distinct in siZe and function from the genetic insula erence. tors of fruit ?y and chicken. The invention provides a recombinant polynucleotide con FIELD OF THE INVENTION taining a plant genetic insulator comprising at least one copy of a polynucleotide having the sequence set forth in SEQ ID The invention relates to the ?eld of plant molecular biology NO:9 (5'GAATATATATATATTC3') or a polynucleotide and biotechnology. Speci?cally, the invention relates to poly region having a sequence that is a variant or fragment of the nucleotide sequences that can minimize or eliminate the posi sequence set forth in SEQ ID N019, Wherein the polynucle tion effect on a transgene in a plant. otide region has a genetic insulator activity. BACKGROUND OF THE INVENTION The invention also provides a vector, comprising: a repli 20 cable vector; and the nucleic acid mentioned above that is Transgenic technology is Widely used in biotechnology. In inserted into the vector. Preferably, the vector is an expression eukaryotes, the temporal and spatial expression of transgenes vector, a plant vector, or a plant expression vector. The inven is regulated by transcription factors through their interaction tion also provides a host cell, in Which the vector is situated. With enhancer elements. HoWever, eukaryotic enhancer-pro The host cell may be a plant cell or a microorganism. moter interactions lack the speci?city for precise temporal 25 The invention further provides a transgenic plant contain and spatial patterns of transgenic expression. The expression ing the genetic insulator polynucleotide. The invention is also of a transgene can be affected by (a) the promoter of a select directed to a recombinant seed containing the genetic insula able marker gene that is closely linked With the transgene, tor polynucleotide. and/or (b) by chromosomal genes that ?ank the transgene, The invention provides a method for expressing a polypep Which is often referred to as the “position effect”. These 30 tide in an organism comprising: constructing a vector com effects are often not desirable, especially When tissue-spe prising the genetic insulator polynucleotide; inserting the ci?c, precisely controlled, or optimal transgene expression is vector into the organism, Wherein the genetic insulator Wanted. sequence is recombined into the organism; and alloWing the It is knoWn that a eukaryotic genome has organizational organism to express the polypeptide. In this method, the properties that rely on the ability of the chromosome to estab 35 polypeptide may be encoded on an insert in the vector. Alter lish autonomous functional units. The polynucleotide natively, the polypeptide may be encoded on the genome of sequences that separate these domains are called genetic insu organism. Further, the described nucleic acid may be inserted lator elements. These genetic insulator elements can buffer a immediately upstream of the nucleic acid encoding the transgene from position effects, so that an introduced trans polypeptide. Preferably, the organism is a plant. More pref gene can be expressed independent of its location in the 40 erably, the plant may be Arabidopsis or tobacco. chromosome. In addition, a genetic insulator may repress nonspeci?c interactions betWeen enhancers and promoters. BRIEF DESCRIPTION OF THE DRAWINGS Thus, it could be possible to obtain precise gene expression by using an appropriate genetic insulator to shield the effects FIG.
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