WO2001007061A1 - Dosage de cellules entieres - Google Patents

Dosage de cellules entieres Download PDF

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Publication number
WO2001007061A1
WO2001007061A1 PCT/US1999/017001 US9917001W WO0107061A1 WO 2001007061 A1 WO2001007061 A1 WO 2001007061A1 US 9917001 W US9917001 W US 9917001W WO 0107061 A1 WO0107061 A1 WO 0107061A1
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WO
WIPO (PCT)
Prior art keywords
streptococcus
cells
gene
group
host cell
Prior art date
Application number
PCT/US1999/017001
Other languages
English (en)
Inventor
Frank Fan
Yinduo Ji
Howard Kallender
Tong Li
Damien Mcdevitt
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to PCT/US1999/017001 priority Critical patent/WO2001007061A1/fr
Publication of WO2001007061A1 publication Critical patent/WO2001007061A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • This invention relates to newly developed methods for discovering a range of therapeutic compounds, particularly antimicrobial compounds and identifying their cellular targets using a whole cell assay It is particularly suited for carrying out therapeutic compound screening assays in bacterial host cells and eukaryotic host cells This invention also relates to compositions of matter useful in carrying out the methods of the invention as well as therapeutic compounds developed using such methods BACKGROUND OF THE INVENTION
  • “Host cell” is a cell which has been transformed or transfected or into which genetic information has been introduced, or which is capable of transformation or transfection or introduction into said cell by an exogenous polynucleotide sequence
  • Isolated means altered “by the hand of man” from its natural state, ( e , if it occurs in nature, it has been changed or removed from its o ⁇ ginal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein
  • Polynucleot ⁇ de(s) generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typicalK double-stranded or triple-stranded regions or a mixture ot single- and double- stranded regions
  • polynucleotide ' as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA The strands in such regions may be from the same molecule or from different molecules The regions may include all of one
  • Polypept ⁇ de(s) refers to any peptide or protein comp ⁇ smg two or more amino acids joined to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins
  • Polypeptides may contain amino acids other than the 20 gene encoded amino acids
  • Polypept ⁇ de(s)” include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acids
  • Va ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any
  • An object ot the invention is a method of screening for therapeutic drugs comprising the steps of providing at least one host cell comprising at least one recombinant regulatable gene, contacting said host cell with at least one candidate compound at one level of induction of gene expression, and detecting altered metabolism in said host cell of the contacting step
  • Another object of the invention is a method of screening for therapeutic drugs comprising the steps of providing at least one host cell comprising at least one recombinant regulatable gene, contacting said host cell in a first contacting step with at least one candidate compound at a first level of induction of gene expression, detecting altered metabolism in said host cell of said first contacting step contacting said host cell in a second contacting step with at least one candidate compound at a second level of induction of gene expression, and detecting altered metabolism in said host cell of said second contacting step
  • a further embodiment of the invention is a method wherein a recombinant gene is on an episomal element or integrated into a chromosome of said host cell
  • Another embodiment is a method wherein the at least one level is two or more levels.
  • a still further embodiment of the invention is a method wherein said at least one recombinant regulatable gene is selected from the group consisting of a Gram positive bacterium, a Gram negative bacterium, a streptococcus, S pneumoniae, a staphylococcus, S aureus, enterococci, Enterocotcus faecahs, Enterococcus faecium, a Bacillus, and Bacillus subtilis
  • said at least one recombinant regulatable gene is selected from the group consisting of hepatic cells, vascular cells, neuronal cells, dermal cells renal cells, pancreatic cells, gut cells, bone cells, muscle cells, transformed cells, and carcinoma cells
  • Another embodiment is a method wherein said altered metabolism comprises an alteration or modulation in viability, growth, proliferation, differentiation, gene expression, gene product activity, lysis, cell division, chemotaxis, motility, cytoskelatel structure or motion, nuclear structure, meiosis. mitosis, translation, transcription, sister chromatid exchange, cell permeability, surface receptors, refractive index, sporulation.
  • a preferred embodiment of the invention is a method wherein said at least one recombinant regulatable gene is selected from the group consisting of a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, M ⁇ cobacter ⁇ um Neisseria, Haemoph ⁇ us, Actinomycetes, Streptonn cetes, Nocardia, Enterobacter, Yersima, Fancisella, Pasturella, Moraxella, Acmetobacter, Erys ⁇ elothri x Branhamella, Actinobacillus, Streptobacillus, Listeria, Ca mmatobacterium, Brucella, Bacillus,
  • Another preferred embodiment of the invention is a method of screening for therapeutic drugs comprising the steps of providing at least one host cell comprising at least one gene under the control of a regulatable promoter, contacting the host cell with at least one candidate compound and detecting altered metabolism in the host cell of the contacting step
  • a method wherein the gene is selected from the group consisting of eubacte ⁇ a or eukaryotes, particularly a human is also provided by the invention.
  • a further embodiment of the invention is a method wherein the altered metabolism comprises inhibition of the encoded protein activity
  • a method is also provided wherein the detecting step further comprises detecting host cell death
  • a method is provided wherein the host cell lacks a complete copy of the gene under the control of its native promoter
  • a method or composition wherein the regulated genes are on episomal element or integrated into a chromosome of the host cell is also provided by the invention.
  • a host cell wherein the gene expression level is regulated is also provided by the invention
  • a polynucleotide comprising a gene expressibly linked to an regulatable promoter BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows a graph demonstrating the putative mechanism of one preferred embodiment of the invention DETAILED DESCRIPTION OF THE INVENTION
  • the natural promoter of the gene is replaced with a heterologous, regulatable promoter (e g , an inducible or repressible promoter) in the chromosome of a gene- expressing microbial host cell, such as by homologous recombination (in a preferred embodiment insertional mutagenesis is used since, for example, it is more rapid than a double crossover and should give the same phenotype
  • a gene construct comprising a regulatable promoter is referred to herein as a "hybrid gene,” ' regulatable gene” or ' recombinant regulatable gene '
  • this method there is an extra copy of the first stretch of base pairs (e g , about 300-700 base pairs, preferably about 500 base pairs) of the gene present, still under the control of the native promoter This is, in a preferred embodiment, not sufficient sequence to encode active gene product
  • inventions include, for example, double crossover mutagenesis to provide efficient and complete promoter replacement, insertional mutagenesis to place a gene plus recombinant promoter in a specific, non-essential locus, and knocking out the chromosomal copy of the gene along with its natural promoter, transforming host cells with a plasmid containing the gene under control of the regulatable promoter, transforming host cells with a plasmid containing the gene under control of the regulatable promoter, and then knocking out the chromosomal copy of the gene plus the natural promoter; transforming host cells with a plasmid containing partial antisense strand of the gene under the control of the regulatable promoter
  • Preferred host cells and other cells useful in the invention include, but are not limited to, any bacteria, and also a member of the genus Streptococcus, Staphylococcus, Bordetella, Corvnebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomvcetes, Nocardia, Enterobacter, Yersima, Fancisella, Pasturella, Moraxella, Acinetobacter, Erys ⁇ elothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobactertum, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rick
  • Streptococcus py ogenes Streptococcus agalactiae, Streptococcus faecahs. Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria memngitidis, Staphylococcus aureus, pa ⁇ cularly Staphxlococcus aureus strain RN4220, Staphylococcus epidermidis, Corvnebacterium diptheriae, Gardnerella vagina s, Mycobacterium tuberculosis.
  • a unicellular or filamentous eukaryote including but not limited to, a protozoan, a fungus, a member of the genus Saccharormces Kluve om ⁇ ces or Candida and a member of the species Saccharomvces ceriMseae Kluveromvces lactis- or Candida albicans
  • a eukaryotic cell, tissue, organ, or organisms especially human cells and tissues, including but not limited to, hepatic cells, vascular cells, neuronal cells, dermal cells, renal cells, pancreatic cells, gut cells, bone cells, muscle cells, transformed cells, and carcinoma cells and
  • Regulatable promoters particularly inducible promoters useful in the invention include, but are not limited to, ⁇ [A P ms tne v/7? repressor gene, from various bacteria, such as Bacillus sp and Lactobacillus pentosus, P[ ac/ ⁇ plus the lacR or lad repressor gene, from various bacteria, such as E coh, S aureus and Lactococcus lacti, hybrid promoters consisting of, for example, an E coh lac repressor/operator and a -10 and -35 region of various promoters, such as phages SPO-1 (known as P S p ac ) and T5, F X y[/ tet - a hybrid consisting of the E co Tn/0 let repressor/operatoi and the Bacillus subtilis xylA -10 and - 35 regions, P-p ⁇ plus the T7 RNA polymerase gene under the control of one of
  • a therapeutic compound screen may be run at both level 1 and level 2 (see Figure 1 ), or at additional levels, and therapeutic compound hits are determined, for example, by their ability to reduce host cell viability or growth or induce lysis, as measured by any method known in the art to detect such changes in the state of a host cell such as, a reduction in the rate of increase of optical density (herein "OD") at 600nm, or other appropriate OD or detection method, at level 1 but not level 2 or some other level
  • OD optical density
  • An alternative preferred embodiment of this screen invention uses a different, preferably more sensitive readout to OD alteration in order to assess therapeutic compounds
  • Therapeutic compound hits are identified by their ability to reduce the reporter output, such as fluorescence or luminescence, among other marker genes and gene products well known in the art
  • the candidate compound may be useful as a therapeutic compound
  • This may be readily determined using any of the many well known methods for testing therapeutic activity, particularly antimicrobial activity, such as, for example, by disk diffusion assay followed by an MIC determination
  • Another application of this technology is for determining the cellular target of therapeutic compounds
  • the natural promoter of the proposed target gene is replaced with a heterologous, regulatable promoter in the chromosome of a gene-expressing host cell, particularly a microbial host cell, as described above Host cells are grown in the presence
  • the method of the invention is formatted for high throughput screening (herein “HTS") Skilled artisans can readily adapt the method of the invention for HTS
  • a particularly preferred embodiment of the screening methods of the invention is a high throughput screen for compounds that interfere with the proper functioning of gene expression or protein
  • Potential therapeutic compounds identified using the method of the invention include, among other things, small organic molecules, polynucleotides, peptides, polypeptides and antibodies that bind host cell polynucleotides or polypeptides, or mimic the activity of a host cell polypeptides
  • Potential antagonists include a small molecule that binds to a host cell polynucleotides or polypeptides thereby preventing binding of natural factors such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules
  • Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules Any molecule from any source can be used as a candidate compound in the methods of the invention, but it is preferred that candidate compounds be small organic molecules
  • kits comprising at least one host cell lacking a host cell gene and the host cell comprising at least one recombinant, regulatable host cell gene of the invention
  • kits comprising at least one host cell lacking a host cell gene and the host cell comprising at least one recombinant, regulatable host cell gene of the invention
  • a further preferred kit comprises a polynucleotide encoding a recombinant host cell gene controlled by an regulatable promoter Kits comprising a host cell gene expressibly linked to an regulatable promoter are also preferred
  • the RAT operon encodes an essential, heterot ⁇ me ⁇ c protein called tRNA-dependent amidotransferase (hence RAT, also known as Glu-tRNA ⁇ m amidotransferase or Glu-AdT - Curnow-AW, et al PNAS 94, 11819-1 1826 (1997))
  • the natural promoter of the RAT operon was replaced with a heterologous, regulatable promoter (Pspac) plus a constitutively expressed lad gene, in the chromosome of S aureus RN4220 by insertional mutagenesis In this way, there is an extra copy of the first stretch of 500 base pairs of the RAT operon present, still under the control of the native promoter This is not sufficient sequence to encode active RAT protein
  • the levels of full length RAT protein could be titrated by varying the level of IPTG inducer between 0 and ImM, showing the same curve as in Figure 1
  • Immunoblot experiments using a RAT-specific polyclonal antibody demonstrated that, even in the absence of inducer, the spac promoter was leaky, such that RAT was expressed above wild type levels
  • a Lad overexpression plasmid pLacI
  • pLacI Lad overexpression plasmid
  • Example 2 Determination of cellular target of a compound using a S. aureus strain with regulatable defl gene.
  • the defl gene in Staphylococcus aureus encodes an essential protein called polypeptide deformylase
  • the natural promoter of the defl single gene operon was replaced with a heterologous, regulatable promoter (Pspac) plus a constitutively expressed lad gene, in the chromosome of 5 aureus RN4220 by insertional mutagenesis
  • Pspac regulatable promoter
  • a constitutively expressed lad gene in the chromosome of 5 aureus RN4220 by insertional mutagenesis
  • the levels of Defl protein could be titrated by varying the level of IPTG inducer between 0 and I mM, showing the same curve as in Figure 1
  • the antimicrobial activity of an inhibitor compound of Defl determined through high throughput screening using an in vitro enzymatic assay, was tested against the regulatable defl strain at various IPTG concentrations Increasing concentrations of the inducer, which led to increasing amount of Defl protein in the cell, resulted in elevation of the MIC values of the compound (Table 1 )
  • a control experiment using nine antibiotics nonspecific to Defl protein did not show variation of MIC values under different inducer concentration Therefore, the antimicrobial activity of the compound is due to the specific inhibition of the polypeptide deformylase
  • This experiment demonstrates an application of the invention using strains with regulatable promoter to identify the cellular targets of antimicrobial compounds
  • Example 3 Do nregulation of a target gene expression in S. aureus using a regulated antisense hla gene.
  • the hla gene encodes alpha-toxin in S. aureus.
  • a 621 bp hla fragment was cloned into pYJ335 downstream of the tetracycline inducible promoter (Ptet/xyl ) in antisense orientation.
  • This shuttle vector carrying antisense hla construct was introduced into S. aureus WCUH29.
  • alpha-toxin could be downregulated after induction transcription of antisense hla RNA using tetracycline or anhydrotetracycline.
  • Induced antisense hla RNA downregulated chromosomally derived hla gene expression in xttr o approximately 14-fold Most importantly, this reduction completely eliminated the lethality of the infection
  • a control S aureus carrying sense hla construct did not show any effect on expression of alpha-toxin in vitr o and on the lethality of the infection at the presence of induction

Abstract

L'invention concerne des méthodes nouvellement développées de découverte de composés thérapeutiques utilisant un système de dosage basé sur des cellules. Cette invention concerne également des compositions de matières utiles dans la mise en application des méthodes de l'invention de même que des composés thérapeutiques développés à l'aide de ces méthodes.
PCT/US1999/017001 1999-07-27 1999-07-27 Dosage de cellules entieres WO2001007061A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
US7815868B1 (en) 2006-02-28 2010-10-19 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US7820427B2 (en) 2001-11-30 2010-10-26 Fluidigm Corporation Microfluidic device and methods of using same
US7833708B2 (en) 2001-04-06 2010-11-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US7887753B2 (en) 2000-11-16 2011-02-15 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US8658418B2 (en) 2002-04-01 2014-02-25 Fluidigm Corporation Microfluidic particle-analysis systems
US8871446B2 (en) 2002-10-02 2014-10-28 California Institute Of Technology Microfluidic nucleic acid analysis
US9714443B2 (en) 2002-09-25 2017-07-25 California Institute Of Technology Microfabricated structure having parallel and orthogonal flow channels controlled by row and column multiplexors
US10131934B2 (en) 2003-04-03 2018-11-20 Fluidigm Corporation Thermal reaction device and method for using the same

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US5646024A (en) * 1986-03-11 1997-07-08 Plant Genetic Systems, N.V. Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
US9926521B2 (en) 2000-06-27 2018-03-27 Fluidigm Corporation Microfluidic particle-analysis systems
US8673645B2 (en) 2000-11-16 2014-03-18 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US10509018B2 (en) 2000-11-16 2019-12-17 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US7887753B2 (en) 2000-11-16 2011-02-15 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
EP2381116A1 (fr) 2000-11-16 2011-10-26 California Institute of Technology Appareil et procédés pour effectuer des analyses et criblage à haut rendement
US8273574B2 (en) 2000-11-16 2012-09-25 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US9176137B2 (en) 2000-11-16 2015-11-03 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US8455258B2 (en) 2000-11-16 2013-06-04 California Insitute Of Technology Apparatus and methods for conducting assays and high throughput screening
US7833708B2 (en) 2001-04-06 2010-11-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US8486636B2 (en) 2001-04-06 2013-07-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US8936764B2 (en) 2001-04-06 2015-01-20 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US8163492B2 (en) 2001-11-30 2012-04-24 Fluidign Corporation Microfluidic device and methods of using same
US9643178B2 (en) 2001-11-30 2017-05-09 Fluidigm Corporation Microfluidic device with reaction sites configured for blind filling
US7820427B2 (en) 2001-11-30 2010-10-26 Fluidigm Corporation Microfluidic device and methods of using same
US8658418B2 (en) 2002-04-01 2014-02-25 Fluidigm Corporation Microfluidic particle-analysis systems
US9714443B2 (en) 2002-09-25 2017-07-25 California Institute Of Technology Microfabricated structure having parallel and orthogonal flow channels controlled by row and column multiplexors
US8871446B2 (en) 2002-10-02 2014-10-28 California Institute Of Technology Microfluidic nucleic acid analysis
US9579650B2 (en) 2002-10-02 2017-02-28 California Institute Of Technology Microfluidic nucleic acid analysis
US10328428B2 (en) 2002-10-02 2019-06-25 California Institute Of Technology Apparatus for preparing cDNA libraries from single cells
US10940473B2 (en) 2002-10-02 2021-03-09 California Institute Of Technology Microfluidic nucleic acid analysis
US10131934B2 (en) 2003-04-03 2018-11-20 Fluidigm Corporation Thermal reaction device and method for using the same
US8420017B2 (en) 2006-02-28 2013-04-16 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US7815868B1 (en) 2006-02-28 2010-10-19 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening

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