Pioneer Hi-bred International, Inc.

University of California

The Johns Hopkins University

Yue Yun

Justin Schares

Randal Arnold

Matthew Paul Cope

Abraham Phillip Lee

Xuan Li

Hemantha Kumar Wickramasinghe

Tza-Huei J Wang

Xinli Emily Wu

Helena Claire Zec

Roberto Barreiro

Clifford Paul Hunter

Aniruddha Kaushik

Kuangwen Hsieh

Do-Hyun Lee

Yinglei Tao

Dohyun Lee

Michelle A Digman

Ning Ma

Lingshu Liu

Chengfeng Zhao

Yi-Fan Hsieh

Brant Axt

Justin Andrew Schares

H Kumar Wickramasinghe

Aniruddha M Kaushik

Kevin Day

Wayne Matthew Crismani

Methods and devices for single cell analysis using fluorescence lifetime imaging microscopy (FLIM) are disclosed. The methods utilize microfluidic devices which use traps to immobilize cells for FLIM analysis. The analysed cells may be sorted before or after imaging and may be plant, animal, or bacterial cells. Analysis of the FLIM data may use a phasor plot and may be used to identify a metabolic pattern of the single cells.

Microfluidic label-free isolation and identification of cells using fluorescence lifetime imaging (FLIM)

Systems and methods for transfection using a microfluidic device are disclosed. Microdroplets encapsulate cells, transfection molecules, and cationic lipid transfection reagent. Droplet chaotic advection in a rendering channel of the system results in a uniform lipid-DNA complex (lipoplex) formation, which can improve gene delivery efficacy. The shear stress exerted on cell membranes during the chaotic mixing increases membrane permeability, which when combined with the co-confinement of cell and lipoplex, improves transfection efficiency of the cell. The systems and methods can be used for a variety of applications such as gene therapy, in vitro fertilization, regenerative medicine, cancer treatment, and vaccines.

Microfluidic systems and methods for lipoplex-mediated cell transfection

The invention provides methods for obtaining genetic material from plant embryos while preserving their viability as well as methods for performing a molecular analysis of plant embryos, particularly with small quantities of genetic material. The methods may include the steps of collecting shed cellular material from one or more embryos; obtaining DNA from the shed cellular material; performing a molecular analysis of the DNA; and germinating at least one of the one or more embryos. A further extension of this method includes determining whether to germinate and grow the embryo or to discard the embryo based on its genotype as part of a breeding process.

Systems and methods for genotyping seed components

Microfluidic systems and methods to generate and analyze microcapsules comprising biological sample, such as for example, single cells, cellular contents, microspore, protoplast, are disclosed. The microcapsules comprising the biological sample can be preserved by a polymerization process that forms a hydrogel around the biological sample. The hydrogel microcapsules can be trapped in a trapping array or collected in an output reservoir and subject to one or more assays. The trapping array or the output reservoir can be disposed over a porous layer that can filter the continuous phase (e.g., oil) in which the microcapsules are dispersed in the microfluidic device. The pores of the porous layer are configured to be smaller than the size of the microcapsules to prevent the flow of the microcapsules through the porous layer.

Systems and methods to encapsulate and preserve organic matter for analysis

A microfluidic system includes a microfluidic chip including a channel layer and a fluid control layer operatively connected to the channel layer, the channel layer having one or more fluid channels. The one or more channels are configured to contain a plurality of droplets. A valve control system is provided to control flow of fluid through the one or more fluid channels in the channel layer. The microfluidic system also includes a droplet impedance detection and feedback control system operatively connected to the valve control system. The droplet impedance detection and feedback control system is configured to detect at least a position of at least one droplet in a fluid channel and to send a signal to the valve control system to operate a particular valve at a particular time based on the detected position of the at least one droplet.

Impedance based feedback control of microfluidic valves

Methods of non-destructively obtaining the genotype of a plant cell from a plant sample are disclosed. The plant cell is isolated from a plant cell sample using an integrated microfluidic device. The integrated microfluidic device includes an individual cell trap located downstream from a microfluidic channel and, the microfluidic device is configured to trap the plant cell in the individual cell trap. mRNA is extracted from the plant cell by contacting the plant cell with an atomic force microscope (AFM) probe and by attracting mRNA from loci of interest to the probe end using a dielectrophoresis DEP force under the alternating current (AC) field applied to the probe. The genotype of the plant cell is determined from cDNA obtained from the extracted mRNA. Alternatively, the mRNA is analyzed to determine gene expression patterns of the plant cell.

Single cell microfluidic device

Methods for obtaining genetic material from plant embryos while preserving their viability are provided. In the methods, preservation of viability may be maintained by suspending the embryos in an aqueous solution surrounded by an oil matrix. Genetic material may be obtained from an aliquot of the aqueous solution and may be used directly for molecular analysis, or whole genome amplification may be performed prior to molecular analysis.

Plant embryo storage and manipulation

Methods for preserving viability of plant tissues such as plant embryos are provided herein. Also included are methods for storing genomic DNA and/or molecular marker assay materials in an oil bilayer as part of a high-throughput molecular characterization system. Moreover, plant embryos may be treated while in an oil matrix. The treatment may include chromosome doubling,-mediated transformation, or herbicide selection as part of an embryo rescue process.

Methods and devices for creating doubled haploid embryos using oil matrices

Methods for obtaining genetic material from plant embryos while preserving their viability as well as methods for performing a molecular analysis of plant embryos, particularly with small quantities of genetic material by collecting shed cellular material from one or more plant embryos; obtaining DNA from the shed cellular material; performing a molecular analysis of the DNA; and germinating at least one of the one or more plant embryos. A further extension includes determining whether to germinate and grow the embryo or to discard the embryo based on its genotype as part of a breeding process. Also provided are methods of genotyping embryos using embryo shed cellular material contained in or on agar and methods of analyzing plant embryonic tissue derived from microspores.

The invention includes methods for obtaining samples of maternal tissue from seeds, obtaining genetic material from the maternal seed tissue, and performing a molecular analysis on the genetic material from the maternal seed tissue to determine maternal lineage of a single seed. The invention also includes methods for establishing a consensus maternal genotype from maternal seed tissues obtained from multiple seeds as well as methods for determining paternal lineage.

Systems and methods for pericarp genotyping

The present application relates to a continuous droplet flow microfluidic system, including a microfluidic chip including an optical detection section; a stage assembly including a microfluidic chip holder configured to receive the microfluidic chip and a plurality of heating elements arranged to heat a plurality of separate sections of the microfluidic chip to a corresponding plurality of different temperatures; and an optical detection system arranged to detect fluorescent light emitted from said optical detection section of the microfluidic chip.

Growing community of inventors

Johnston, IA, United States of America

Yue Yun