Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-08-14
2002-11-12
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S468000, C435S285100
Reexamination Certificate
active
06479288
ABSTRACT:
This invention relates to a method for introducing an agent into the cytosol or plasma membrane of a cell using a lipid-coated tip of a pipette or rod or the like to contact, but not penetrate, the cell. In addition, the invention relates to a pipette or rod having a lipid-coated tip for introducing agents into the cytosol or plasma membrane of a cell and also to a method of coating a tip of a pipette or rod with the lipid.
The micro-injection of agents such as proteins and nucleic acid (mRNA and DNA) into the cytosol of living cells is a powerful technique which has led to many new discoveries. Furthermore, it underlies some new approaches to molecular and genetic engineering and is becoming useful in in vitro fertilisation. However, micro-injection by the insertion of a glass micro-pipette into the cell is a potentially damaging event and is limited, at present, to cells which are both large and robust. Micro-injection of small mammalian cells (spherical diameter =2-15 &mgr;m) or very flat cells (1-2 &mgr;m thickness) has always been difficult to achieve without damaging the cell and resulting in poor cell survival. The penetration of small cells, especially spherical cells, requires a rapid entry and exit “stab”, as slow withdrawal of the micropipette often results in breaking the cell. Thus “stab injection”, where the micro-injector is within the cell for approximately 100 msec, high pressure (100-200 mbar) has to be used to introduce sufficient material into the cell during that time. This must be carefully controlled as insufficient pressure results in too little material being injected and excessive pressure causes cell damage or rupture. The cell must also be firmly attached to a substrate for the “stab”to effectively penetrate the cell membrane. Apart from these problems, the process is essentially blind and, because of the small size of the cell and the large volume of the cell cytoplasm occurred by organelles such as the nucleus, lysosomes and ER, insertion of the micro-injector at high speed is likely to cause intracellular damage. For example, the micro-pipette entering the cell with a velocity of approximately 700 &mgr;m/sec is likely to displace, damage or enter the nucleus (which in small cells such as neutrophils and basophils accounts for up to 50% of the cell volume) rather than enter directly into the cytosol even if the plasma membrane survives.
Traditional pressure injection has been successfully used in a number of cell-types, including, hepatocytes (Cobbold) and Jurkat cells. However, the success rate is limited, with many cells failing to survive stab injection. There have been various attempts to minimise the problem of cell damage, including the use of pharmacological Ca
2+
channel blockers. Other techniques have also been used for introducing agents, such as peptide inhibitors, antibodies etc into cells, by membrane permeabilisation using chemical or biological molecules, or techniques such as by electroporation. While this approach can be useful, cell damage is great, and mechanisms by which the cell repairs the damage are poorly understood, but which may involve Ca
2+
and active signalling by the cell. Thus the recovered cells may not represent those in a state that was originally intended for study. Another method which has been used is internal perfusion. In this technique, the micro-pipette does not enter the cell, but membrane is sucked into the mouth of the micro-pipette causing a local rupture of the cell membrane at that point. This minimises damage to intracellular structures, but the process of “breaking into” the cell results in an irreversible seal and the cell must remain attached to the pipette for its survival. This precludes studies of cell shape change etc, and the negative pressure required to break-in also withdraws cytosol if not carefully controlled. In any case, during the period that the micro-pipette is attached diffusion of material both from the micro-pipette into the cell and from the cytosol into the micro-pipette occurs. Diffusion out of the cell of important small molecules such as GTP, ATP etc essentially limits the usefulness of this technique to those processes in which they are not involved or are regulated by the contents of the micro-pipette. All these techniques, while useful, thus have limitations.
Other approaches which do not involve glass micro-injection have also been used. One early approach was the use of lipid fusion, either by the use of liposomes or erythrocyte ghost function. While lipid fusion can be successful for introducing cDNA etc into cells as with lipofection, the amount of material injected is very small and perhaps only one copy of the cDNA is sufficient for the experiment. For example to inject a small cell (d=10 &mgr;m) with 1% of its volume by fusion of liposomes (d=50 nm) requires thousands of fusion events. This number of fusion events is unlikely and the use of liposomes for cell physiology has not been widely successful. Fusion of larger vesicles such as red cell ghosts has also been used, where one fusion event can introduce a large amount of material. However, a large amount of foreign membrane is also introduced and the cells are essentially hybrids rather than original cells.
According to a first aspect of the present invention, there is provided a method for introducing an agent in to the cytosol and/or plasma membrane of a cell, comprising the steps of:
(a) coating at least a portion of a tip of a transferring apparatus with a lipid;
(b) bringing the lipid-coated tip of the transferring apparatus into contact with the cell; and
(c) transferring at least some of the contents of the transferring apparatus into the cytosol and/or plasma membrane of the cell without entering the cytoplasm.
The transferring apparatus is preferably a pipette or rod. The pipette is usually a micro-pipette (usually manufactured of glass) and the rod is usually a micro-rod. The micro-rod is a device (usually made of glass) which is solid and has no internal bore. A micro-pipette may be used for lipid-assisted micro-injection of substances into the cytosol or into the plasma membrane, whereas a micro-rod may be used to introduce substances exclusively into the plasma membrane. The micro-rod has the advantage that lipids and lipid-soluble molecules such as proteins can be transferred from the micro-rod to the cell and vice versa without aqueous transfer into the cytosol. In the case of a rod, the “contents” of the transferring apparatus may be present in the lipid on the tip.
It has been found that the technique provides fusion between the lipid at the tip and the cell membrane which results in a channel into the cell cytosol, without the possibility of intracellular organelle damage, and low pressure in the transferring apparatus ensures that the amount of material injected is controlled and does not unduly damage the cell. Furthermore, as only contact, rather than penetration, is required for agent introduction with this approach, the cell need not be firmly adherent. The applicants have carried out the use of simple lipid assisted micro-injection (“SLAM”) on human neutrophils, when loosely adherent as spherical individual cells or spread on glass coverslips with a cell thickness of just 1-3 &mgr;m. These cells have been very difficult, if not impossible, to undergo successful micro-injection by conventional means.
Preferably, at least some of the contents of the transferring apparatus are transferred under pressure, which pressure is small enough to prevent damage to the cell contents. This pressure may typically be in the range of between 5 and 40 mbar.
The method is particularly useful when the cell is a living cell which remains living after transferring the contents of the transferring apparatus into the cytosol and/or plasma membrane of the cell. For example, the cell may be a small mammalian cell. Thus, the method of the invention has particular use in introducing agents in to a cell having a spherical diameter of between 2 and 15 &mgr;m, or a cell of substantiall
Hallett Maurice Bartlett
Laffafian Iraj
McKelvey Terry
University of Wales College of Medicine
Young & Thompson
LandOfFree
Method and apparatus for introducing substances into the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and apparatus for introducing substances into the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and apparatus for introducing substances into the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2964765