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Fluorescence activated cell sorting (FACS) enrichment to generate plants

A plant cell, plant technology, applied in the field of fluorescence activated cell sorting

Inactive Publication Date: 2015-06-24
DOW AGROSCIENCES LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has some inefficiencies due to the sensitivity of the assay, so there is still room for improvement

Method used

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  • Fluorescence activated cell sorting (FACS) enrichment to generate plants
  • Fluorescence activated cell sorting (FACS) enrichment to generate plants
  • Fluorescence activated cell sorting (FACS) enrichment to generate plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Identification of paralogous FAD2 and FAD3 target sequences from bacterial artificial chromosome libraries

[0090] BAC construction

[0091] Bacterial artificial chromosome (BAC) libraries were purchased from a supplier (Amplicon Express, Pullman, WA). The BAC library consisted of 110,592 BAC clones containing high molecular weight genomic DNA (gDNA) fragments isolated from Brassica napus var. DH10275. Digest gDNA with BamHI or HindIII restriction enzymes. The isolated gDNA fragment of approximately 135 Kbp was ligated into pCC1BAC vector (Epicentre, Madison, WI) and transformed into E. coli strain DH10B (Invitrogen). The BAC library consisted of an even number of BAC clones constructed with two different restriction enzymes. Therefore, the BAC library constructed by HindIII consisted of 144 384-well plates. Similarly, the BAC library constructed by BamHI consisted of 144 384-well plates. A total of 110,592 BAC clones were isolated and arrayed into 28...

Embodiment 2

[0123] Example 2: Design of a Zinc Finger Binding Domain Specific to the FAD2 Gene

[0124]Zinc finger proteins were designed against DNA sequences encoding various functional sequences of the FAD2 gene locus as previously described. See, eg, Urnov et al. (2005) Nature 435:646-651. Exemplary target sequences and recognition helices are shown in Tables 6 and 8 (recognition helix region design) and Tables 7 and 9 (target sites). In Tables 8 and 9, nucleotides in the target site that are in contact with the ZFP recognition helix are indicated in capital letters; non-contacting nucleotides are indicated in lower case. Zinc finger nuclease (ZFN) target sites were designed to bind 5 target sites of FAD2A and 7 target sites of FAD3. The FAD2 and FAD3 zinc fingers were designed to be incorporated into a zinc finger expression vector encoding a protein having at least one finger with a CCHC structure. See US Patent Publication No. 2008 / 0182332. In particular, the last finger in eac...

Embodiment 3

[0141] Example 3: Evaluation of Zinc Finger Nuclease Cleavage of the FAD2 Gene

[0142] construct assembly

[0143] Plasmid vectors containing ZFN expression constructs for exemplary zinc finger nucleases identified using yeast assays as described in Example 2 were designed and completed using skills and techniques generally known in the art. Each zinc finger coding sequence was fused to a sequence encoding the opaque-2 nuclear localization signal (Maddaloni et al. (1989) Nuc. Acids Res. 17(18):7532), upstream of the zinc finger nuclease.

[0144] Next, the opaque-2 nuclear localization signal::zinc finger nuclease fusion sequence is paired with the complementary opaque-2 nuclear localization signal::zinc finger nuclease fusion sequence. Thus, each construct consisted of a single open reading frame consisting of the 2A sequence from the genus spp. (beta tetrasomy) virus (Mattion et al. (1996) J. Virol. 70:8124- 8127) separated by two opaque-2 nuclear localization signal::z...

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Abstract

An Engineered Transgene Integration Platform (ETIP) is described that can be inserted randomly or at targeted locations in plant genomes to facilitate rapid selection and detection of a GOI that is perfectly targeted (both the 3' and 5' ends) at the ETIP genomic location. One element in the invention is the introduction of specific double stranded breaks within the ETIP. In some embodiments, an ETIP is described using zinc finger nuclease binding sites, but may utilize other targeting technologies such as meganucleases, TALs, CRISPRs, or leucine zippers. Also described are compositions of, and methods for producing, transgenic plants wherein the donor or payload DNA expresses one or more products of an exogenous nucleic acid sequence (e.g. protein or RNA) that has been stably-integrated into an ETIP in a plant cell. In embodiments, the ETIP facilitates testing of gene candidates and plant expression vectors from ideation through Development phases.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application Serial No. 61 / 697,890, filed September 7, 2012. field of invention [0003] The present disclosure relates to the field of fluorescence activated cell sorting for generating plants. In a preferred embodiment, the present disclosure describes FACS enrichment of edited, regenerable protoplasts for generation of fertile, edited plants. Background of the invention [0004] The fluorescence activated cell sorter (FACS) was invented by Bonner, Sweet, Hulett, Herzenberg, and others in the late 1960's for cell sorting and flow cytometry of viable cells. The Becton Dickinson immunocytometry system was introduced as a commercial machine in the early 1970's. Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method to sort a heterogeneous mixture of biological cells into two or more containers, one cell at a tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12Q1/24A01H5/00
CPCC12N15/8212C12N15/8213C12N15/8241
Inventor G·斯潘根伯格S·萨哈布J·梅森
Owner DOW AGROSCIENCES LLC
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