Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-04-30
2003-05-13
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S173500, C435S375000, C435S285100
Reexamination Certificate
active
06562623
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 a kit for introducing a substance into a cell.
The introduction of foreign substances, such as foreign DNA, into cells is termed transfection. This technique 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 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.
In Animal Cell Culture: A Practical Approach, (edited by R. I. Freshney, Second Ed., 1992, page 56-57, IRL Press, OUP) a method of oxygenating a cell culture is discussed. The method involves sparging. This is the bubbling of gas through a liquid medium comprising the culture. The reference states that gas bubbles having a high surface energy may damage animal cell membranes if not properly controlled.
An object of the present invention is to overcome the above drawbacks and to provide an efficient method of transfection. Accordingly, the present invention provides a method for introducing a substance into a cell, which method comprises:
(a) generating bubbles of a gas in a liquid medium comprising the cell, the bubbles being capable of forming a hole in the surface of the cell when one or more bubbles interact with the cell; and
(b) introducing the substance into the cell.
In the present invention the bubbles are capable of forming a hole in the surface of the cell. This means that the dimensions of the bubbles, and their composition (in terms of both the liquid medium and the gas of the bubbles) are sufficient to enable the bubbles to form holes in the cell surface.
In the present invention, any method can be used to generate the bubbles of gas in the liquid medium containing the cells, provided that the dimensions of the bubbles are controlled such that the bubbles are capable of forming transient holes in the cell (in particular when interacting with the cell surface). The formation of holes in the cell surface using bubbles is termed aeroporation in the context of the present application. Preferably, the dimensions of the bubbles are comparable to the dimensions of the cell. For example, a preferred bubble radius ranges from approximately one third times the radius of the cell to five times the radius of the cell.
In one embodiment, the bubbles of gas are generated by introducing a gas into the liquid medium in a sparging process. Typically, but not exclusively, sparging involves the passage of gas through a porous element (e.g. a filter) which is in contact with the liquid medium. The porous element ensures that the gas enters the liquid evenly across a given area of the liquid, thus forming small bubbles extending evenly throughout the volume of the liquid.
In another embodiment, the bubbles of gas may be generated by a chemical, or an electrochemical reaction, such as by the decomposition of hydrogen peroxide contained in the liquid medium.
In a further embodiment, the bubbles of gas may be generated in the liquid medium by a depressurisation process. Depressurisation typically involves reducing the pressure to which the liquid medium is exposed, such that the solubility of the dissolved gas is reduced, causing the formation of bubbles in the liquid. Without being bound by theory, it is believed that the cells in the liquid medium act as nuclei for the formation of the bubbles of gas. This embodiment thereby advantageously allows the formation of bubbles in close proximity with the cells, increasing the efficiency of transfection.
The dissolved gas may be gas that is present in the liquid medium naturally under ambient conditions, or in an alternative embodiment gas may be dissolved in the liquid medium prior to carrying out the present method. Gas may be dissolved in the liquid medium by any means, such as by controlling the temperature and/or pressure, or preferably by increasing the pressure. The type of gas used is not especially limited, but it is preferred that the gas used is air, or another gas that is soluble in an aqueous medium. The rate of generation of the bubbles of gas and the size of the bubbles may be controlled by varying the rate and extent of the decrease of the pressure.
The extent of foam formation on the surface of the liquid is preferably controlled by introducing a foam-controlling agent. The foam controlling agent used in the present invention is not especially limited and may include any known foam controlling agent, such as a commercially available fatty acid.
In the present methods, without being bound by theory, it is believed that gas bubbles having a sufficiently small radius have sufficient surface energy (or surface tension) that on interacting with the cells (such as contacting the cells and in particular, bursting when in contact with or in close proximity to the cells) a hole is formed in the cell surface. The hole in the cell surface may be a rip or tear in the cell membrane and may also include a hole formed by entirely removing a portion of the cell membrane. The hole provides access to the inside of the cell, allowing the substance to enter the internal parts of the cell, such as the cytoplasm.
There is no significant decrease in the viability of the cells, even though holes are formed in the cell surface. The holes formed in the cells are transient, r
Carmody & Torrance LLP
Immunoporation LTD
Ketter James
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