DK2802656T3 - Cellulær indkapsling med høj gennemstrømning til screening eller udvælgelse - Google Patents
Cellulær indkapsling med høj gennemstrømning til screening eller udvælgelse Download PDFInfo
- Publication number
- DK2802656T3 DK2802656T3 DK13700541.9T DK13700541T DK2802656T3 DK 2802656 T3 DK2802656 T3 DK 2802656T3 DK 13700541 T DK13700541 T DK 13700541T DK 2802656 T3 DK2802656 T3 DK 2802656T3
- Authority
- DK
- Denmark
- Prior art keywords
- cells
- selection
- steps
- solubilized
- cell
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Virology (AREA)
- Ecology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Claims (15)
1. Fremgangsmåde til udvælgelse af et sekvenssæt fra et bibliotek af ek-sprimerede nukleinsyresekvenser, hvor - der tilvejebringes en flerhed af celler, hvor hver celle omfatter en eksprime-ret nukleinsyresekvens eksprimeret som et targetprotein i cellen, - flerheden af celler indkapsles i et indkapslingstrin, omfattende ° at behandle flerheden af celler med et kationisk polysaccharid i et kationisk behandlingstrin, o at behandle flerheden af celler med et anionisk polysaccharid i et anionisk behandlingstrin, - hvor der ved indkapslingstrinnet opstår en flerhed af indkapslede celler, - at solubilisere membranen af de indkapslede celler i et solubiliseringstrin, hvorved der opstår en flerhed af solubiliserede kamre, - at bringe flerheden af solubiliserede kamre, i et markeringstrin, i kontakt med o en ligand til targetproteinet, hvor liganden bærer en detekterbar label, eller med o en indikator for en enzymatisk aktivitet af targetproteinet, hvor den enzymatiske aktivitet omdanner indikatoren til en detekterbar label, - at udvælge et undersæt af flerheden af solubiliserede kamre som en funktion af detekterbar label, der forekommer i de solubiliserede kamre i et udvælgelsestrin, hvorved der opstår en udvælgelse, og - at isolere de eksprimerede nukleinsyresekvenser fra udvælgelsen som et udvalgt sekvenssæt i et isoleringstrin.
2. Fremgangsmåde ifølge krav 1, hvor det kationiske polysaccharid er chito-san, og/eller det anioniske polysaccharid er alginat eller hyaluronsyre.
3. Fremgangsmåde ifølge et af de foregående krav, hvor det kationiske behandlingstrin kommer før det anioniske behandlingstrin.
4. Fremgangsmåde ifølge et af de foregående krav, hvor en sekvens af trin omfattende et kationisk behandlingstrin efterfulgt af et anionisk behandlings- trin gentages to til ti gange.
5. Fremgangsmåde ifølge et af de foregående krav, hvor den detekterbare label er et fluorescerende farvestof.
6. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor liganden er et oligopeptid, en agonist, antagonist, et substrat eller en overgangstilstandsanalog, som binder til en variant af targetproteinet.
7. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor det udvalgte sekvenssæt overføres til en flerhed af celler og underkastes en sekvens af indkapslingstrin, solubiliseringstrin, markeringstrin, udvælgelsestrin og isoleringstrin.
8. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor det udvalgte sekvenssæt efter isoleringstrinnet diversificeres ved a. forstærkning af de eksprimerede nukleinsyresekvenser ved hjælp af en fremgangsmåde, hvor mutationer indføres i den forstærkede sekvens, og/eller ved b. deletion eller insertion af sekvenstrakter i de eksprimerede nukleinsyresekvenser, og efterfølgende, det udvalgte sekvenssæt underkastes en anden sekvens af indkapslingstrin, solubiliseringstrin, markeringstrin, udvælgelsestrin og isoleringstrin.
9. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor biblioteket af eksprimerede nukleinsyresekvenser er et bibliotek af homologe sekvenser med mindst 60 % identitet med hinanden.
10. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor den eksprimerede nukleinsyresekvens er indeholdt i et transgen-ekspressionskonstrukt.
11. Fremgangsmåde ifølge krav 10, hvor transgen-ekspressionskonstruktet er et plasmid.
12. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor targetproteinet er et G-protein-koblet receptorprotein, en ion kanal, et enzym, en kernereceptor, en transkriptionsfaktor eller et DNA/RNA-bindende protein.
13. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor solubilise-ringstrinnet fuldføres ved at udsætte de indkapslede celler for et rensemiddel.
14. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor cellerne er bakterieceller.
15. Fremgangsmåde ifølge et af de ovenfor nævnte trin, hvor den detekterba-re label er et fluorescerende farvestof og udvælgelsestrinnet fuldføres ved en fluorescerende cellesortering.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP12150453.4A EP2612916A1 (en) | 2012-01-09 | 2012-01-09 | Cellular high throughput encapsulation for screening or selection |
| PCT/EP2013/050330 WO2013104686A1 (en) | 2012-01-09 | 2013-01-09 | Cellular high throughput encapsulation for screening or selection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DK2802656T3 true DK2802656T3 (da) | 2017-01-16 |
Family
ID=47563484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK13700541.9T DK2802656T3 (da) | 2012-01-09 | 2013-01-09 | Cellulær indkapsling med høj gennemstrømning til screening eller udvælgelse |
Country Status (12)
| Country | Link |
|---|---|
| US (2) | US10870846B2 (da) |
| EP (2) | EP2612916A1 (da) |
| JP (1) | JP6189329B2 (da) |
| CN (1) | CN104093838B (da) |
| AU (1) | AU2013208951B2 (da) |
| CA (1) | CA2860852C (da) |
| DK (1) | DK2802656T3 (da) |
| ES (1) | ES2611736T3 (da) |
| HK (1) | HK1199061A1 (da) |
| HU (1) | HUE032889T2 (da) |
| PL (1) | PL2802656T3 (da) |
| WO (1) | WO2013104686A1 (da) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2612916A1 (en) | 2012-01-09 | 2013-07-10 | Universität Zürich | Cellular high throughput encapsulation for screening or selection |
| CN107075545A (zh) | 2014-07-30 | 2017-08-18 | 哈佛学院院长及董事 | 探针文库构建 |
| CA2971111C (en) * | 2014-12-22 | 2023-05-23 | Universitat Zurich | Directed evolution of membrane proteins in eukaryotic cells with a cell wall |
| EP3635159A4 (en) * | 2017-05-11 | 2021-01-13 | The Florey Institute of Neuroscience and Mental Health | ENCAPSULATION OF EUKARYOT CELLS FOR EXPRESSED SEQUENCE CELL SCREENING |
| US11788123B2 (en) | 2017-05-26 | 2023-10-17 | President And Fellows Of Harvard College | Systems and methods for high-throughput image-based screening |
| WO2020150789A1 (en) * | 2019-01-25 | 2020-07-30 | The Australian National University | Encapsulated cells |
| EP4061937A1 (en) | 2019-11-20 | 2022-09-28 | LeadXpro AG | Method of enabling pooled-library based nucleic acid constructs screening |
| WO2021216789A1 (en) * | 2020-04-21 | 2021-10-28 | University Of Maryland, College Park | System, device, and method for single-cell encapsulation and culture |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69228247T2 (de) * | 1991-08-10 | 1999-07-08 | Medical Res Council | Behandlung von Zellpopulationen |
| US20040241759A1 (en) * | 1997-06-16 | 2004-12-02 | Eileen Tozer | High throughput screening of libraries |
| JP5291279B2 (ja) | 2000-09-08 | 2013-09-18 | ウニヴェルジテート・チューリッヒ | 反復モジュールを含む反復タンパク質の集合体 |
| GB0802474D0 (en) * | 2008-02-11 | 2008-03-19 | Heptares Therapeutics Ltd | Mutant proteins and methods for selecting them |
| GB0918564D0 (en) * | 2009-10-22 | 2009-12-09 | Plasticell Ltd | Nested cell encapsulation |
| EP2612916A1 (en) | 2012-01-09 | 2013-07-10 | Universität Zürich | Cellular high throughput encapsulation for screening or selection |
-
2012
- 2012-01-09 EP EP12150453.4A patent/EP2612916A1/en not_active Withdrawn
-
2013
- 2013-01-09 AU AU2013208951A patent/AU2013208951B2/en active Active
- 2013-01-09 DK DK13700541.9T patent/DK2802656T3/da active
- 2013-01-09 WO PCT/EP2013/050330 patent/WO2013104686A1/en active Application Filing
- 2013-01-09 ES ES13700541.9T patent/ES2611736T3/es active Active
- 2013-01-09 EP EP13700541.9A patent/EP2802656B1/en active Active
- 2013-01-09 CA CA2860852A patent/CA2860852C/en active Active
- 2013-01-09 JP JP2014551604A patent/JP6189329B2/ja active Active
- 2013-01-09 PL PL13700541T patent/PL2802656T3/pl unknown
- 2013-01-09 US US14/371,031 patent/US10870846B2/en active Active
- 2013-01-09 HU HUE13700541A patent/HUE032889T2/hu unknown
- 2013-01-09 CN CN201380005074.8A patent/CN104093838B/zh active Active
-
2014
- 2014-12-12 HK HK14112550.1A patent/HK1199061A1/zh unknown
-
2020
- 2020-11-18 US US16/951,457 patent/US11661676B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| HUE032889T2 (hu) | 2017-11-28 |
| EP2802656B1 (en) | 2016-10-19 |
| JP2015503926A (ja) | 2015-02-05 |
| JP6189329B2 (ja) | 2017-08-30 |
| US11661676B2 (en) | 2023-05-30 |
| CA2860852A1 (en) | 2013-07-18 |
| CN104093838A (zh) | 2014-10-08 |
| ES2611736T3 (es) | 2017-05-10 |
| CN104093838B (zh) | 2016-09-07 |
| PL2802656T3 (pl) | 2017-05-31 |
| WO2013104686A1 (en) | 2013-07-18 |
| EP2802656A1 (en) | 2014-11-19 |
| CA2860852C (en) | 2021-01-12 |
| HK1199061A1 (zh) | 2015-06-19 |
| US20210309992A1 (en) | 2021-10-07 |
| AU2013208951A1 (en) | 2014-08-14 |
| US20150031549A1 (en) | 2015-01-29 |
| EP2612916A1 (en) | 2013-07-10 |
| US10870846B2 (en) | 2020-12-22 |
| AU2013208951B2 (en) | 2018-02-22 |
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