Method for introducing nucleic acids into higher eukaryotic cells
AssigneeBoehringer Ingelheim International FmbH
InventorsMichael Buschle, Ernst Wagner, Wolfgang Zauner
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Abstract
Process and medium for the transfection of higher eukaryotic cells with DNA/polycation complexes, wherein a medium is used which contains ethyleneglycol and/or glycerol. The medium may also contain a substance which prevents the acidification of the endosomes, and/or a lower alcohol such as ethanol. The process is particularly suitable for the transfection of primary cells such as fibroblasts. Stably transformed cells can be obtained thereby, e.g. tumour cells for use as tumour vaccines.
Claims49
A method for introducing nucleic acids/polycation complexes into animal cells, said method comprising adding the complexes to the cells in the presence of a chemical selected from the group consisting of (a) ethyleneglycol, (b) glycerol, and (c) ethyleneglycol and glycerol.
A method for preparing cells which express a heterologous polypeptide from a stably transfected DNA molecule, the method comprising:
- (a) transfecting cells in the presence of a chemical selected from the group consisting of (i) glycerol ethyleneglycol, and (iii) glycerol and ethyleneglycol with a complex of a DNA molecule coding for the heterologous polypeptide and a polycation;
- (b) cultivating the cells, and
- (c) selecting stable cell clones.
The method according to claim 28, wherein the cells are tumor cells.
A medium for transfection of animal cells, the medium comprising:
- (a) nutrients and conventional additives, as the active components;
- (b) complexes of nucleic acid and a polycation which is selected from the group consisting of (i) nonconjugated polycation (ii) polycation fully conjugated with an internalizing factor of the cells, and (iii) polycation partially conjugated with an internalizing factor of the cells, and
- (c) a chemical selected from the group consisting of (i') ethyleneglycol (ii') glycerol, and (iii') ethyleneglycol and glycerol.
Dependencies are inferred from the claim text.