Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-02-09
2002-02-05
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S459000, C530S387100, C530S388100, C530S388220, C530S389100, C530S391100
Reexamination Certificate
active
06344357
ABSTRACT:
The present invention relates to a method for introducing a substance into a cell, and in particular to an efficient transfection method involving a low incidence of cell-death. The invention also relates to kits for introducing a substance into a cell.
Throughout this text, the introduction of foreign substances, such as nucleic acid protein, peptides or other biological molecules into cells is termed transfection. Transfection, particularly of genetic material, has recently proved to be one of the most important techniques in molecular biology, particularly in relation to genetic engineering and protein engineering. The technique has allowed foreign DNA to be expressed in cells. This is of scientific interest in studying gene transcription and has a wide range of commercial applications involving expressing commercially useful gene products in convenient types of cell. More recently there has been interest in introducing both proteins and drugs into living cells without damaging the cells. A significant problem to be overcome when developing such techniques is the general imperviousness of the cell membrane. The cell membrane is normally impervious to even small molecules, unless they are very lipophilic. Even short-term damage to the cell membrane to render it more permeable tends to result in cell-death. This is a particular problem associated with electroporation, discussed below.
A number of methods have been devised for transfecting cells with foreign DNA or other substances. Early methods involved binding DNA to particles such as diethylaminoethyl (DEAE) cellulose or hydroxyapatite and adding pre-treated cells which are capable of taking up particles containing DNA. These early methods are very inefficient, the level of transfection achievable being very low.
More recently methods have been developed which make use of liposomes loaded with DNA that can be fused with cells. A further technique involves subjecting cells, typically plant cells, to an electric shock which causes the formation of holes in the cells. This method is termed electroporation.
In Biotechniques, Vol 17 No. 6 1994, page 118-1125, Clarke et al. disclose a method for introducing dyes, proteins and plasmid DNA into cells using an impact-mediated procedure. In this method, compressed gas is used to propel glass beads dispersed as a uniform aerosol onto adherent cells growing on a culture substratum. The impact of beads on the cells creates plasma membrane wounds. Molecules such as dyes, proteins and plasmid DNAs diffuse from the extracellular environment directly into the cytoplasmic compartment of the cell through the wounds.
In
Nucleic Acids Research
, Vol. 18, No. 21, 1990, p.6464, the effect of the osmolarity of the transfection medium is studied in relation to electroporation methods. It was reported that the optimum osmolarity of the transfection medium for transfection by electroporation is around 300 mOsm.
A significant problem associated with the above treatments is that they are very inefficient. In addition, a large proportion of the cells are killed by the above treatments. Moreover, the treatments are not selective. In fact, no methods are presently available for the selective transfection of cells. Furthermore, in the method of Clarke et al, only a limited number of cells can be transfected in a single treatment.
An object of the present invention is to overcome the above drawbacks and to provide an efficient method of transfection with good cell survival rates and the possibility of selective transfection of a sub-population of cells in a sample. Accordingly, the present invention provides a method for introducing a substance into a cell, which method comprises:
(a) contacting the cell with a recognition agent to bind the recognition agent to a recognition site on the surface of the cell; and
(b) separating the recognition agent from the cell thereby forming a hole in the surface of the cell.
The method of the invention is conveniently referred to herein, as “immunoporation”, although not all methods of the invention actually involve any immunological components or interactions.
The cell can be contacted with the substance to be introduced simultaneously with the formation of the hole in step (b), or alternatively in a subsequent separate step. Introduction of the substance may thus occur as a result of contact of the substance with the cell which contains one or more holes which facilitate passage of the substance into the cell. This said hole allows passage into the cell membrane or more preferably the cytoplasm of the cell.
The hole formed is typically transient in that it only exists for a very short period before the cell membrane again forms a substantially continuous layer. The important characteristic of the hole is that it enables the substance which it is intended to introduce into the cell to pass from the outside to the inside of the cell.
The invention will now be described in further detail by way of example only, with reference to the accompanying drawings, in which:
FIG. 1
shows the effect of the osmolarity of the transfection medium on the transfection of HL60 cells;
FIG. 2
shows confocal images of (A) promyelocytic HL-60 cells transfected with TMR-dextran using anti-CD71-coated Dynafect beads, (B) promyelocytic HL-60 cells transfected with TMR-dextran using anti-CD11b-coated Dynafect beads (negative control), (C) 3 day DMSO differentiated HL-60 cells transfected with TMR-dextran using anti-CD71-coated Dynafect beads (negative control), (D) 3 day DMSO differentiated HL-60 cells transfected with TMR-dextran using anti-CD11b-coated Dynafect beads; and
FIG. 3
shows FACS analysis of the expression of GFP in: (A) promyelocytic HL-60 cells transfected with pEGFP-C1 using anti-CD71-coated Dynafect beads, (B) HL-60 cells transfected with pEGFP-C1 using anti-CD71-coated Dynafect beads 1 day after induction with DMSO, (C) HL-60 cells transfected with pEGFP-C1 using anti-CD71-coated Dynafect beads 3 days after induction with DMSO, (D) promyelocytic HL-60 cells transfected with pEGFP-C1 using anti-CD11b-coated Dynafect beads, (E) HL-60 cells transfected with pEGFP-C1 using anti-CD11b-coated Dynafect beads 1 day after induction with DMSO and (F) HL-60 cells transfected with pEGFP-C1 using anti-CD11b-coated Dynafect beads 3 days after induction with DMSO.
The step (b) of the present method is preferably carried out in a liquid medium. In the present context this is termed an immunoporation medium. To maximise the efficiency and rate of transfection, the liquid medium used to transfect the cells typically has an osmolarity of from 0.1-4.0 times the osmolarity of the cells. In the context of the present invention, the osmolarity of the cell means the normal osmolarity of the untreated cell. Preferably, the liquid medium has an osmolarity of from 30-1200 mOsm. When the cells to be transfected are adherent, the osmolarity of the liquid medium is preferably less than the osmolarity of the cells, more preferably from 30-150, e.g. 40-100 mOsm and most preferably from 40-50 mOsm. When the cells to be transfected are in the form of a suspension, the osmolarity of the liquid medium is preferably greater than the osmolarity of the cells, more preferably from 700-1100 mOsm, more preferably still from 800-1000 mOsm and most preferably from 950-1000 mOsm. Thus non-isotonic conditions, relative to the osmolarity of the cells, allows for most efficient immunoporation and the liquid medium will therefore preferably not be isotonic or approximately isotonic having regard to the osmolarity of the cells.
The osmolarity of a liquid medium is a measure of the concentration of ions in the medium. The ions present in the immunoporation medium are not limited to a particular type of ion, provided that they do not inhibit transfection and can be tolerated by the cells. An immunoporation medium having the desired osmolarity may be formulated using 10 times concentrated Earl's balanced salt solution (EBSS) (Earl, W. R., 1934, Arch. Exp. Zell. Forsch., Vol. 16, p. 116) containing nutrient factors as a b
Carmody & Torrance LLP
Immunoporation LTD
Ketter James
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