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Zinc finger nuclease-mediated homologous recombination

A technology of homologous recombination and nuclease, applied in the direction of recombinant DNA technology, hydrolase, peptide, etc., can solve the problems that cannot be widely used

Active Publication Date: 2009-09-09
DOW AGROSCIENCES LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since meganuclease recognition sites are rare (or non-existent) in typical plant genomes, insertion of suitable meganuclease recognition sites is hampered by the same difficulties associated with other genomic alterations, Therefore, these methods cannot be widely applied

Method used

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  • Zinc finger nuclease-mediated homologous recombination
  • Zinc finger nuclease-mediated homologous recombination
  • Zinc finger nuclease-mediated homologous recombination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0353]Example 1 - Design and Generation of Targeting Vectors

[0354] A. Overall Structure of the Target Sequence

[0355] Target constructs for tobacco (dicots) include the following 7 building blocks, such as figure 1 Shown: i) hygromycin phosphotransferase (HPT) expression cassette, which comprises the drive by Agrobacterium tumefaciens (A.tumifaciens) open reading frame-24 (orf-24) 3' untranslated region (UTR) (Gelvin etc., 1987, EP222493) the Escherichia coli HPT gene (Waldron et al., 1985, Plant Mol.Biol.18:189-200) terminated Arabidopsis (A.thaliana) ubiquitin-3 (ubi-3) promoter (Callis et al., 1990, J.Bio.Chem.265:12486-12493); ii) homologous sequence-1, which comprises the matrix attachment region (MAR) of tobacco (N. tabacum) RB7 (Thompson et al., 1997, WO9727207) ; iii) 5' green fluorescent protein (GFP) gene fragment (Evrogen Joint Stock Company, Moscow, Russia), which is expressed by the modified Agrobacterium tumefaciens mannopine synthase (Δmas) promoter (Pe...

Embodiment 2

[0374] Example 2 - Generation of transgenic cell lines with integrated target sequences

[0375] Two different suspension cultures of tobacco cells in which the target sequence of Example 1 was stably integrated via Agrobacterium transformation were used. The first culture, called NT1, was obtained from Arnold Bendich (University of Washington, Seattle, WA, USA). The culture proliferated to 15-20 μ diameter cells in 20-30 cell clusters with a doubling time of approximately 48 hours. NT1 cell suspension cultures were maintained in media containing the following components: MS basal salts (PhytoTechnology Labs M524), 137.4 mg / L K 2 HPO 4 , 30g / L sucrose, 2.22mg / L2,4-D, 1mg / L thiamine hydrochloride, 100mg / L myo-inositol and 0.5g / L MES, pH 5.7. NT1 cells were subcultured every 7 days by adding 40 mL of fresh MS-based medium to 1 mL of packed cell volume (PCV).

[0376] The second tobacco cell culture used (which was called BY2) was obtained from Jun Ueki (Japan Tobacco, Iwata,...

Embodiment 3

[0381] Example 3 - Screening and Characterization of Targeted Transgenic Events

[0382] Hygromycin resistance transgenic events (as described in Example 2) generated from transformation of targeting vectors into BY2 or NT1 tobacco cell cultures were analyzed as follows.

[0383] Initial analyzes to screen for these transgenic events included GUS expression analysis to demonstrate target sequence accessibility, PCR analysis of partial and full-length target sequences to confirm the presence and integrity of the target vector, and Southern blot analysis to determine integrated copy number of the target sequence. A subset of transgenic events showing GUS expression contained a single copy of the full-length target sequence; they were selected for reconstitution of suspension cultures to generate target lines for subsequent retransformation. These reconstructed target lines also underwent further characterization, which included more comprehensive Southern blot analysis, sequenc...

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Abstract

The present invention discloses methods and compositions for targeted integration of an exogenous sequence into a predetermined target site in a plant genome.

Description

field of invention [0001] The present disclosure is in the fields of genome engineering, gene targeting, targeted chromosomal integration, and protein expression in plants. Background of the invention [0002] A major area of ​​interest in agriculture, especially in light of the determination of the full nucleotide sequence of a large number of plant genomes, is the targeted alteration of the genome sequence. In particular, the ability to switch endogenous plant sequences would facilitate numerous applications such as, for example, optimizing crop traits affecting nutritional value, yield, stress tolerance, pathogen resistance, and agrochemical resistance and / or adapting plants for use as biofactories To produce pharmaceutical compounds or industrial chemicals. [0003] In eukaryotes, attempts have been made to alter the genome sequence in cultured cells by exploiting the natural phenomenon of homologous recombination. See, eg, Capecchi (1989) Science 244:1288-1292; US Pat...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/82
CPCC12N15/8213C12N9/22C12N15/65A01H1/06C12N5/14C07K14/00C07K2319/81
Inventor 蔡其华杰弗里·米勒威廉·M·安利罗比·J·加里森约瑟夫·F·皮托利诺贝丝·C·鲁宾-威尔逊莉萨·L·舒伦伯格安德鲁·F·沃登
Owner DOW AGROSCIENCES LLC
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