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US20020006664A1 - Arrayed transfection method and uses related thereto - Google Patents

Arrayed transfection method and uses related thereto Download PDF

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Publication number
US20020006664A1
US20020006664A1 US09/817,003 US81700301A US2002006664A1 US 20020006664 A1 US20020006664 A1 US 20020006664A1 US 81700301 A US81700301 A US 81700301A US 2002006664 A1 US2002006664 A1 US 2002006664A1
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Prior art keywords
dna
cells
mixture
interest
gelatin
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US09/817,003
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David Sabatini
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Whitehead Institute for Biomedical Research
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Individual
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Priority to US09/817,003 priority Critical patent/US20020006664A1/en
Application filed by Individual filed Critical Individual
Publication of US20020006664A1 publication Critical patent/US20020006664A1/en
Assigned to WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH reassignment WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SABATINI, DAVID M.
Priority to JP2002575306A priority patent/JP2004530426A/ja
Priority to EP02725351A priority patent/EP1379642A4/fr
Priority to CA002440378A priority patent/CA2440378A1/fr
Priority to PCT/US2002/009265 priority patent/WO2002077264A2/fr
Priority to US10/379,130 priority patent/US6951757B2/en
Priority to US10/403,720 priority patent/US20030203486A1/en
Priority to US10/403,630 priority patent/US20030228601A1/en
Assigned to SILICON VALLEY BANK DBA SILICON VALLEY EAST reassignment SILICON VALLEY BANK DBA SILICON VALLEY EAST SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AKCELI, INC.
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the resulting product (a surface bearing DNA and plated cells) is maintained under conditions that result in entry of DNA into plated cells, thus producing an array (a surface bearing an array) of reverse transfected cells that contain defined DNA and are in discrete, defined locations on the array.
  • arrays are the subject of this invention.
  • FIG. 4A is a schematic for making transfected cell microarrays using a well-less transfection of plasmid DNAs in defined areas of a lawn of mammalian cells. Plasmid DNA dissolved in an aqueous gelatin solution is printed on a glass slide using a robotic arrayer. The slide is dried and the printed array covered with a lipid transfection reagent. After removal of the lipid, the slide is placed in a culture dish and covered with cells in media. The transfected cell microarray forms in 1-2 days and is then ready for downstream assays. An alternative method in which the lipid is added to the DNA/gelatin solution prior to printing is also described.
  • FIG. 5D show two examples of the morphological phenotypes detectable in the transfected cell microarrays described in FIG. 5C. Clusters shown are E8 and F7 from array 2.
  • Detection of effects on recipient cells can be carried out by a variety of known techniques, such as immunofluorescence, in which a fluorescently labeled antibody that binds a protein of interest (e.g., a protein thought to be encoded by a reverse transfected DNA or a protein whose expression or function is altered through the action of the reverse transfected DNA) is used to determine if the protein is present in cells grown on the DNA spots.
  • a protein of interest e.g., a protein thought to be encoded by a reverse transfected DNA or a protein whose expression or function is altered through the action of the reverse transfected DNA
  • feature refers to an area of a substrate having a homogenous collection of a target sequence (or sequences in the case of certain co-transfection embodiments).
  • One feature is different than another feature if the target sequences of the different features have different nucleotide sequences.
  • recombinant cells include any cells that have been modified by the introduction of heterologous nucleic acid.
  • Control cells include cells that are substantially identical to the recombinant cells, but do not express one or more of the proteins encoded by the heterologous nucleic acid.
  • cell surface receptor refers to molecules that occur on the surface of cells, interact with the extracellular environment, and transmit or transduce the information regarding the environment intracellularly in a manner that may modulate intracellular second messanger activities or transcription of specific promoters, resulting in transcription of specific genes.
  • Co-transfections can be performed with transfected cell microarrays if the solution spotted on the surface where reverse transfection occurs contains more than one plasmid or nucleic acid construct.
  • the co-transfection features can include, for example, 2-10 different target sequences per feature, 10-100 different target sequences per feature, or even more than 100 different target sequences per feature.
  • the compound would be attached to the solid support via the photocleavable linker and the tag is attached through a catechol ether linker via carbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem 59:4723-4724).
  • This orthogonal attachment strategy permits the FACS sorting of the cell/bead entities and subsequent decoding by ECGC after oxidative detachment of the tag sets from isolated beads.
  • the beads can be tagged with two or more fluorescently active molecules, and the identity of the bead is defined by the ratio of the various fluorophores.
  • the host cells are plated (placed) onto the surface bearing the transfection array in sufficient density and under appropriate conditions for introduction/entry of the nucleic acid into the cells.
  • the host cells in an appropriate medium
  • the density of cells can be from about 0.3 ⁇ 10 5 /cm 2 to about 3 ⁇ 10 5 /cm 2 , and in specific embodiments, is from about 0.5 ⁇ 10 5 /cm to about 2 ⁇ 10 5 /cm 2 and about 0.5 ⁇ 10 5 /cm 2 to about 1 ⁇ 10 5 /cm 2 .
  • the appropriate conditions for introduction/entry of DNA into cells will vary depending on the quantity of cells used.
  • a heterologous reporter gene construct can be used to provide the function of an indicator gene.
  • Reporter gene constructs are prepared by operatively linking a reporter gene with at least one transcriptional regulatory element. If only one transcriptional regulatory element is included it must be a regulatable promoter. At least one the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the selected cell-surface receptor whereby activity of the receptor can be monitored via transcription of the reporter genes.
  • Selectivity tests could also be performed on the metabolites of a drug candidate.
  • a radiolabeled drug could be reacted with the appropriate biotransformation agent, such as a liver extract, tissue culture system, or living organism such as a rodent or dog.
  • the radiolabeled metabolites could then be extracted and purified and tested for binding with the array. Metabolites with binding activity could then be characterized further by standard methods.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US09/817,003 1999-09-17 2001-03-22 Arrayed transfection method and uses related thereto Abandoned US20020006664A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US09/817,003 US20020006664A1 (en) 1999-09-17 2001-03-22 Arrayed transfection method and uses related thereto
PCT/US2002/009265 WO2002077264A2 (fr) 2001-03-22 2002-03-22 Procede de transfection en reseau et utilisation associee
JP2002575306A JP2004530426A (ja) 2001-03-22 2002-03-22 アレイ化トランスフェクション法および前記方法に関連する使用
CA002440378A CA2440378A1 (fr) 2001-03-22 2002-03-22 Procede de transfection en reseau et utilisation associee
EP02725351A EP1379642A4 (fr) 2001-03-22 2002-03-22 Procede de transfection en reseau et utilisation associee
US10/379,130 US6951757B2 (en) 1999-09-17 2003-03-04 Transfection method and uses related thereto
US10/403,720 US20030203486A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto
US10/403,630 US20030228601A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US15473799P 1999-09-17 1999-09-17
US19358000P 2000-03-30 2000-03-30
US09/817,003 US20020006664A1 (en) 1999-09-17 2001-03-22 Arrayed transfection method and uses related thereto

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/664,297 Continuation-In-Part US6544790B1 (en) 1999-09-17 2000-09-18 Reverse transfection method

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US10/379,130 Continuation US6951757B2 (en) 1999-09-17 2003-03-04 Transfection method and uses related thereto
US10/403,630 Continuation US20030228601A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto
US10/403,720 Continuation US20030203486A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto

Publications (1)

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US20020006664A1 true US20020006664A1 (en) 2002-01-17

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US09/817,003 Abandoned US20020006664A1 (en) 1999-09-17 2001-03-22 Arrayed transfection method and uses related thereto
US10/379,130 Expired - Lifetime US6951757B2 (en) 1999-09-17 2003-03-04 Transfection method and uses related thereto
US10/403,630 Abandoned US20030228601A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto
US10/403,720 Abandoned US20030203486A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto

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US10/379,130 Expired - Lifetime US6951757B2 (en) 1999-09-17 2003-03-04 Transfection method and uses related thereto
US10/403,630 Abandoned US20030228601A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto
US10/403,720 Abandoned US20030203486A1 (en) 1999-09-17 2003-03-28 Transfection method and uses related thereto

Country Status (5)

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US (4) US20020006664A1 (fr)
EP (1) EP1379642A4 (fr)
JP (1) JP2004530426A (fr)
CA (1) CA2440378A1 (fr)
WO (1) WO2002077264A2 (fr)

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US20030228694A1 (en) 2003-12-11
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US6951757B2 (en) 2005-10-04
JP2004530426A (ja) 2004-10-07
EP1379642A4 (fr) 2006-04-19
CA2440378A1 (fr) 2002-10-03
WO2002077264A3 (fr) 2003-02-20
US20030203486A1 (en) 2003-10-30

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