EP1141340A1 - A method for introducing substances into cells, and use of said method - Google Patents
A method for introducing substances into cells, and use of said methodInfo
- Publication number
- EP1141340A1 EP1141340A1 EP00902243A EP00902243A EP1141340A1 EP 1141340 A1 EP1141340 A1 EP 1141340A1 EP 00902243 A EP00902243 A EP 00902243A EP 00902243 A EP00902243 A EP 00902243A EP 1141340 A1 EP1141340 A1 EP 1141340A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cell
- cells
- protein
- substance
- nucleic acid
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
Definitions
- Newborn neurons in the granule cell layer express markers of differentiated neurons and have morphological characteristics corresponding to differentiated granulae cells (Kaplan, M. S. and Bell, D. H., J. Neurosci. 4: 1429-1441 (1984); Cameron, H. A., Woolley, C. S., McEwen, B. S., and Gould, E., Neuroscience 56: 337-344 (1993);
- the invention is based on the use of this nucleic acid transport system in progenitor cells and stem cells for different purposes. According to the present invention, it is possible to transfer DNA without the help or aid of e.g. viral vectors.
- the invention provides new methods to isolate progenitor cells and stem cells in vivo and in vitro. This isolation may be based on the expression from plasmid containing cDNA of a protein that enables selective identification and isolation based on immunoreactivity, or on the expression by DNA of a protein that enables selective identification and isolation based on the expression of fluorescent proteins, including FACS sorting.
- the invention also provides new methods to transport different substances with e.g. pharmaceutical effects into progenitor cells and/or stem cells.
- nucleic acids may either be used for their ability to make it possible to identify and thus isolate progenitor cells and stem cells from other cells, or for their pharmaceutical effects.
- the method according to the invention may be performed both in vitro, e.g. in a tissue or cell culture, and in vivo.
- the cells into which the substance is transported are preferably cells in the central nervous system.
- the method is especially suitable for the identification of progenitor cells and stem cells.
- the methods according to the invention are used for the purpose of identification it is preferable that the substance that is to be introduced into said cells gives rise to a detectable signal or to a peptide or protein that enables selective identification of stem cells and progenitor cells. Said peptide or pro- tein may then in its turn give rise to a detectable signal, as the case is for e.g. a fluorescent protein, or a marker protein.
- suitable markers for stem cells or progenitor cells are protein components of the transport system, such as receptors and carriers.
- the de- tectable signal may also be obtained by the use of tagged substances, such as a radioactively tagged nucleic acid. It is especially interesting to be able to identify, and thereafter isolate, progenitor cells and stem cells in samples constituted of e.g. different structures of brain tissue taken out of a patient or cells cultured from a patient.
- the in vivo method can be used in order to identify, and subsequently isolate, cells in vivo, in a way similar to the in vitro method described above.
- the method is performed in vivo, it is possible to identify, and thus isolate, stem cells and progenitor cells in different structures of the intact brain
- stem cells and progenitor cells are also possible to propagate stem cells and progenitor cells with the methods according to the inven- tion. This propagation can be performed both in vitro and in vivo.
- the cells which optionally first may have been identified and isolated with the methods according to the invention, are then brought in contact with a substance that comprises or gives rise to peptide or protein that, once it is taken up by the cells, activate proliferation and/or differentiation and/or lineage determination of said cells.
- Figure 1A is a fluorescence photomicrograph showing the result of incubation of progenitor cells in medium with 50 ⁇ g/ml of a plasmid containing the cDNA for GFP;
- Figure IB a lightmicroscopic image showing the same result as figure 1A;
- Figure 1C is a fluorescence photomicrograph showing the result of incubation of progenitor cells in me- dium with 50 ⁇ g/ml of another plasmid not containing the GFP gene;
- Figure ID a lightmicroscopic image showing the same result as figure IB.
- the cells were incu- bated with plasmids containing the cDNA for GFP, and plasmids deficient of the GFP gene, respectively, in a humid atmosphere at 37°C with 5% C0 2 and 95% air for 10 minutes.
- the cells were cultured for 48 h, following DNA exposition. Thereafter the expression of the fluorescent protein was detected using an inverted Leica DMIRB microscope equipped for fluorescence microscopy.
- the cells were viewed in the microscope using excitation of GFP at 488 nm using an Ar-ion laser (Spectra Physics model 2025-05, Sunnyvale, CA) .
- the laser light was sent through a 488- line interference filter followed by a spinning disk to break the coherence and scatter the laser light.
- the laser was collected by a lens and sent through a fluores- cein filter cube (Leica 1-3) into the objective to excite the fluorophores .
- the resulting fluorescence was collected by the same objective and the image was detected by a 3 -chip color CCD-camera (Panasonic) and recorded at 25 Hz frame collection rate by a Super VHS (Panasonic SVHS AG-5700) .
- the CCD images were digitized from tape and processed for presentation.
- Figure 1A is a fluorescence photomicrograph showing this result
- Figure IB shows the respective lightmicroscopic image.
- kidney-derived Cos-7 cells that were incubated in medium with 50 ⁇ g/ml of a plasmid containing the cDNA for GFP for 10 minutes without addition of chemicals that facilitate uptake or transport of DNA, and thereafter grown for 48h before detection, lack expression of green fluorescent protein (GFP) . Detection and experimental procedures for this experiment was iden- tical to that for progenitor cells exposed to plasmid containing the cDNA for GFP.
- GFP green fluorescent protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Neurosurgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Neurology (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE9900134 | 1999-01-15 | ||
SE9900134A SE9900134D0 (en) | 1999-01-15 | 1999-01-15 | A method for introducing substances into cells, and using said method |
PCT/SE2000/000073 WO2000042202A1 (en) | 1999-01-15 | 2000-01-14 | A method for introducing substances into cells, and use of said method |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1141340A1 true EP1141340A1 (en) | 2001-10-10 |
Family
ID=20414133
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00902243A Withdrawn EP1141340A1 (en) | 1999-01-15 | 2000-01-14 | A method for introducing substances into cells, and use of said method |
Country Status (10)
Country | Link |
---|---|
EP (1) | EP1141340A1 (en) |
JP (1) | JP2002534126A (en) |
CN (1) | CN1179047C (en) |
AU (1) | AU751342B2 (en) |
CA (1) | CA2359349A1 (en) |
IL (1) | IL144104A0 (en) |
NZ (1) | NZ513502A (en) |
SE (1) | SE9900134D0 (en) |
WO (1) | WO2000042202A1 (en) |
ZA (1) | ZA200105749B (en) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5750376A (en) * | 1991-07-08 | 1998-05-12 | Neurospheres Holdings Ltd. | In vitro growth and proliferation of genetically modified multipotent neural stem cells and their progeny |
SG54115A1 (en) * | 1993-04-27 | 1998-11-16 | Gerber Scient Products Inc | Thermal printing apparatus with improved power supply |
WO1996015811A1 (en) * | 1994-11-17 | 1996-05-30 | Imperial College Of Science, Technology & Medicine | Internalisation of dna, using conjugates of poly-l-lysine and an integrin receptor ligand |
US5753506A (en) * | 1996-05-23 | 1998-05-19 | Cns Stem Cell Technology, Inc. | Isolation propagation and directed differentiation of stem cells from embryonic and adult central nervous system of mammals |
-
1999
- 1999-01-15 SE SE9900134A patent/SE9900134D0/en unknown
-
2000
- 2000-01-14 JP JP2000593759A patent/JP2002534126A/en active Pending
- 2000-01-14 WO PCT/SE2000/000073 patent/WO2000042202A1/en not_active Application Discontinuation
- 2000-01-14 EP EP00902243A patent/EP1141340A1/en not_active Withdrawn
- 2000-01-14 IL IL14410400A patent/IL144104A0/en unknown
- 2000-01-14 AU AU23372/00A patent/AU751342B2/en not_active Ceased
- 2000-01-14 CN CNB00802698XA patent/CN1179047C/en not_active Expired - Fee Related
- 2000-01-14 NZ NZ513502A patent/NZ513502A/en unknown
- 2000-01-14 CA CA002359349A patent/CA2359349A1/en not_active Abandoned
-
2001
- 2001-07-12 ZA ZA200105749A patent/ZA200105749B/en unknown
Non-Patent Citations (1)
Title |
---|
See references of WO0042202A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2000042202A1 (en) | 2000-07-20 |
NZ513502A (en) | 2003-03-28 |
AU751342B2 (en) | 2002-08-15 |
CN1336959A (en) | 2002-02-20 |
CN1179047C (en) | 2004-12-08 |
ZA200105749B (en) | 2004-01-12 |
JP2002534126A (en) | 2002-10-15 |
AU2337200A (en) | 2000-08-01 |
SE9900134D0 (en) | 1999-01-15 |
IL144104A0 (en) | 2002-05-23 |
CA2359349A1 (en) | 2000-07-20 |
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Owner name: CELLARTIS AB |
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Effective date: 20041119 |