Resistencia del suelo

The majority of the data presented in this thesis was obtained from studies performed on immortalised eukaryotic cell lines. In this section details of the methods of culturing are given for cell lines used.

The HEK293 cell line.

The HEK293 cell line (obtained from Dr L Y Jan, UCSF) is an immortalised human cell line derived from primary embryonic kidney cells transformed with sheared adenovirus type 5 DNA (Graham et. al., 1977).

Growth conditions.

HEK293 cells were cultured in Minimum Essential Medium (MEM) with Earles’ salts and L-Glutamine (Gibco Life Technologies, Paisley, UK) supplemented with 10% Foetal Bovine Serum (of European origin, Gibco Life Technologies, Paisley, UK) and 1% Penicillin-Streptomycin (from a stock of 10,000 units/ml penicillin and 1 mg/ml streptomycin, Gibco Life Technologies, Paisley, UK). Cells were grown at 37°C in a humidified atmosphere of 95% air/5% COj adhered to the surface of tissue culture flasks treated with vacuum gas plasma (Falcon products, Becton Dickinson, Franklin Lakes, New Jersey, USA).

Method o f subculture and maintenance.

Cells were typically subcultured when at approximately 90% confluence. The culture medium was removed and the cells washed twice with Ca^^, Mg^^ free

Dulbeccos’ phosphate buffered saline (Gibco Life Technologies, Paisley, UK). Cells were detached from the culture surface of the flask by addition of 0.25% Trypsin in Dulbeccos’ phosphate buffered saline followed by a 1-2 minute incubation at room temperature. Trypsin was diluted in phosphate buffered saline from a 2.5% (w/v)

stock in saline solution (Gibco Life Technologies, Paisley. UK). After the cells had detached an excess of culture medium containing serum was added to inhibit the tryptic activity. The cell suspension was centrifuged at 340g for 3 minutes. Pelleted cells were resuspended in fresh culture medium at a density of 1.5x10^ viable cells/ml.

An aliquot of 7.5x10^ cells was subsequently used to inoculate another flask. The doubling time of HEK293 cells was typically 24-36 hours in log phase growth and as a consequence subculturing was done once weekly. The culture medium on HEK293 cells was generally removed and replaced with fresh medium once every 3-4 days.

Production/revival o f frozen stocks

Frozen stocks were produced by subculturing 80-100% confluent cells as described above but after centrifugation the cells were resuspended in culture medium containing 10% steriledimethyl sulphoxide (Sigma, Poole, UK) at a density of 1.5x10^ cells/ml. Cells were aliquoted into suitable vials for cryogenic storage in 1ml aliquots before being gradually cooled at 4°C for 30 minutes, -20°C for 30 minutes and -80°C for 30 minutes prior to being transferred to liquid nitrogen for long-term storage. Cells were revived by rapid thawing of an aliquot at 37°C before inoculation of a flask containing fresh pre-warmed culture medium. Cells were incubated under normal conditions for between 6 and 18 hours before culture medium was removed and replaced with fresh culture medium.

The HepG2 cell line.

The HepG2 cell line (obtained from Dr G Bellingham, Department of Medicine, UCL) is a permanent cell line derived from a human hepatocellular carcinoma (Knowles et. al., 1980).

Methods o f subculture, maintenance and production/revival o f frozen stocks.

Cells were subcultured in the same manner described for HEK293 cells. The doubling time of HepG2 cells was 12-24 hours in log phase growth and therefore cells were subcultured twice weekly. The culture medium on HepG2 cells was removed and replaced with fresh medium every 1 to 2 days. The procedure for the production and revival of frozen stocks was the same as the one employed for HEK293 cells.

The 9E10 Hybridoma cell line.

The 9E10 cell line (obtained from Dr S Moss, UCL) is a mouse hybridoma cell line that secretes a monoclonal antibody recognising the amino acid sequence EQKLISEEDL that is part of the carboxy-terminal domain of human c-myc, a transcriptional activator.

Growth conditions

9E10 cells were cultured in identical growth medium and under identical conditions to those used for HEK293 cells. The cells were grown both adhered to tissue culture flasks and in suspension culture using spinner bottles to ensure adequate aeration of the cells.

Method o f subculture

Cells grown adhered to tissue culture flasks were subcultured twice weekly. As 9E10 cells are semi-adherent they were subcultured when at 50% confluence (at confluence > 50% cells detached from the growth surface). The culture medium was removed and replaced with fresh medium. The cells were removed from the surface of the flask by gentle tituration. The cell suspension was then used to inoculate another flask containing fresh medium at a 1:10 dilution. Suspension cultures were initiated with a 1:10 dilution of cell suspension from a 50% confluent flask and could

be maintained for 2-3 months in suspension before a new culture was initiated with fresh cells. Cells in suspension culture were subcultured at a 1:10 dilution once weekly by removal of culture medium and replacement with fresh medium. When growing cells in suspension for subsequent harvest of monoclonal antibody from the supernatant, cells were grown for 2-3 weeks without subculture with twice weekly additions of fresh culture medium.

Production/revival o f frozen stocks.

Frozen stocks of 9E10 cells were produced from 50% confluent adherent cultures. Cells were removed from the growth surface in the manner described above and pelleted by centrifugation at 340g for 3 minutes before resuspension in fresh culture medium containing 10% ^imetliyl sulphoxide (DMSO). Cells were gradually cooled and frozen in liquid nitrogen for long term storage as described in the earlier section. Frozen 9E10 cells were revived in the same manner as HEK293 cells.

Counting viable cells using the Trypan Blue exclusion assay.

Viable cells were counted using a trypan blue exclusion assay in conjunction with a haemocytometer. A 500pl aliquot of cell suspension was removed and mixed with 500pl 0.4% (w/v) Trypan Blue staining solution (Sigma, Poole, UK). A small amount of this suspension was added to the central well of a haemocytometer (Neubauer 0.100mm depth), a coverslip applied and the two chambers allowed to fill by capillary action. The haemocytometer was then observed under the microscope and the number of viable cells in each 1mm square was counted and the mean value taken. Non-viable cells were classified as those that had taken up the Trypan Blue stain. Given that the volume of the 1mm square is 0.1 mm^ or 10 ‘^ml (the depth is stated as 0.100mm), the number of cells per ml can be calculated using the expression;

No. of cells/ml = Mean count/mm^ x 10,000 x 2

where 2 is the dilution factor of the cell suspension in Trypan Blue solution.

2.3 Transfections.

To allow the study of K^tp channel subunits in heterologous systems such as the HEK293 cell line it is necessary to introduce plasmid DNA encoding the

appropriate protein into the cells where it can direct over-expression of the protein of interest. The process of introducing plasmid (or any other form of foreign) DNA into a cell line is known as transfection and this section will outline the principles of transfection and also discuss the method of transfection employed for experiments described in this thesis.

There are several methods that can be used to transfect plasmid DNA into a cell line. The most commonly used methods are calcium phosphate co-precipitation (Graham and Van der Eb, 1973) and liposome mediated gene transfer (Itani et. al., 1987). Other methods of transfection mediated by DEAE-Dextran (McCutchan and Pagano, 1968), Polybrene (Kawai and Nishizawa, 1984) and bacterial protoplasts (Schaffher, 1980) can also be used. All of these methods involve the formation of a complex between DNA and the mediator of the transfection. This complex can ftise with the plasma membrane of the cell and the DNA enters the cytoplasm. Once in the cytoplasm the DNA is trafficked to the nucleus and proteins(s) encoded by the DNA can be expressed using the transcriptional and translational machinery of the cell. The schematic diagram in Figure 2.6 illustrates the principle. Details of plasmid structure were given in Figure 2.1.

Other methods exist for directly introducing plasmid DNA into a cell, without formation of a plasmid-mediator complex. These include electroporation (Potter et. al., 1984) in which a high voltage pulse reversibly permeabilises the cell membrane allowing DNA to enter, scrape loading (Fechhiemer, 1987) where cells are stimulated to take up DNA by mechanically injuring them and microinjection of DNA directly into the nucleus of the cell (Capecchi, 1990). A relatively new technique involves firing plasmid DNA coated with gold particles directly into the cell using a “gene gun”. DNA can also be introduced into cells by infecting them with recombinant vaccinia viruses (Karschin et. al., 1992).

In the studies described in this thesis all transfections were done using a lipid based transfection method. This method was chosen because efficient transfection could be achieved with relatively small quantities of DNA (typically between 0.5 and 1.5p,g) and the methods are simple and well characterised. The experimental

procedure is described in the next section.

Liposome based transfection procedure

Cells were subcultured as described in Section 2.2 and seeded into tissue culture plates with wells of 35mm diameter (Falcon products, Becton Dickinson, Franklin Lakes, New Jersey, USA) at a cell density of 2x10^ cells per well. The cells were subsequently incubated overnight under normal conditions to allow them to adhere to the growth surface and to recover from trypsinisation.

Lipid-plasmid DNA complexes were prepared just prior to transfection as follows; for each transfection 5pi of 2mg/ml Lipofectamine (Gibco Life

Technologies, Paisley, UK), a liposome formulation of cationic and neutral lipids (a 3:1 w/w ratio of the polycationic lipid DOSPA and the neutral lipid DOPE in sterile water), preincubated for 10 minutes in lOOpl Optimem serum free medium (Gibco Life Technologies, Paisley, UK) was added to a tube containing 0.8-1.5pg of total plasmid DNA in lOOpl Optimem. The lipid-DNA mixture was incubated for 30 minutes at room temperature to allow lipid-DNA complex formation. After the incubation period the volume of the transfection mixture was adjusted to 1ml with Optimem. Cells were briefly washed with Optimem to remove contaminating serum proteins before the transfection mixture was added to the cells. Charged proteins present in foetal bovine serum may interfere with the transfection process by

sequestering lipid-DNA complexes and preventing fusion with the target cells. The cells were incubated with the transfection mixture under otherwise normal growth conditions for 5 hours before the transfection mixture was removed and replaced with normal culture medium.

Transfected cells were incubated for 24 hours prior to either selection for stably transfected cells or experiments on transiently transfected cells (the difference between transient and stably transfected cells is explained in Section 2.4).

Cationic lipids in serum free medium Plasmid DNA in serum free mediiun Lipid-DNA complex

6 o 0

0 0

A

0 0

o

+

o o

I

Release of DNA into cell Lipid-DNA complex fusion with cell

Figure 2.6 The principle o f lipid based transfections.

The diagram above shows how a mixture o f cationic and neutral lipids can be used to introduce foreign D N A into mammalian cells. Complexes between positively cliarged Upids and negatively charged D N A can be formed which can then fuse to the plasma membrane o f tlie target cell, facilitating the uptake o f D N A into the cell by an endocytic process (the exact mechanism is unclear). Serum-free medium is used throughout the transfection process as serum proteins can interfere with com plex formation. The same principle o f com plex formation applies to other methods o f transfection using cationic facilitators such as DEAE and polybrene.