Generation of new cell lines

Cells used for transfection were either CHO-K1 cells or a clonal cell line of CHO

cells already expressing the CRE-SPAP (CS) reporter gene construct (CHO-CS

cells). These cells were cultured in T25 flasks to be able to use a lower amount

of DNA and the transfection agent lipofectamine.

Transfection of cells to generate a stable mixed population cell

line

The DNA used in transfections was a circular eukaryotic expression plasmid

(pcDNA3.1) consisting of the desired receptor (e.g. human 1-adrenoceptor)

and an antibody resistance gene (e.g. neomycin). The DNA was in solution (in

double-distilled H2O) and the concentration determined using a UV

spectrophotometer (see Molecular biology: Maxi-prep). The transfection

agent used was lipofectamine, a lipophilic agent that coats the DNA, making it

more membrane permeable to facilitate the uptake of the DNA. Two

solutions were prepared:

Solution A = 3 µg DNA + OPTIMEM (to make up a total of 300 µL)

Solution B = 30 µL lipofectamine + 270 µL OPTIMEM

The two solutions were combined (= transfection mix; total volume 600 µL)

and left at room temperature for 1.5 hours.

After 1 hour, the growth medium on cells that were grown to 60-70 %

confluence in a T25 flask, was replaced with 3 mL OPTIMEM medium and the

cells were incubated for 30 minutes at 37 °C in a 5 % CO2/95 % air atmosphere.

incubated at 37 °C in a 5 % CO2/95 % air atmosphere for 24 h to allow the

uptake of DNA by the cells. The next day the OPTIMEM media containing the

transfection mix was removed and the cells were washed by the addition and

removal of 10 mL DPBS to remove any cells that died during the transfection

process, and replaced with 10 mL of fresh growth media to allow the cells

that survived to recover. Cells that recovered from the transfection process

would start to express the new proteins and thus, the selection process was

started the next day.

Selection for the transfected cells was started 24-48 hours after transfection

by replacing the growth medium with growth medium supplemented with the

relevant antibiotic (e.g. 1 mg/mL geneticin (G418) or 500 µg/mL zeocin;

=selection medium). Successfully transfected cells will express the desired

receptor and the antibiotic resistance gene. Antibiotic resistance allows the

cell to survive the cytotoxic effects of a given antibiotic. This treatment

caused death of cells not expressing the transfected construct. In addition,

cells that have not incorporated the transfected construct into the nuclear

DNA of the cell, but only transiently (e.g. in the cytoplasm) expressed the

antibiotic resistance protein, would also not survive the antibiotic treatment

past a few division cycles. Thus, only cells that have incorporated the

transfected construct into their nuclear DNA have the means for survival.

However, the insertion of the external DNA is a random process and may

disrupt DNA sequences essential to the survival and growth of the cell, in

in which the transfected construct was inserted into an inactive region of the

DNA, thus resulting in very low (or no) transcription of the introduced

sequences. Every couple of days, the selection medium was replaced with

fresh selection medium to remove cells that had died during the selection

process. After 1-2 weeks, the cells were passaged and placed into a new T25

flask. When the rate of cell death declined and the cell population grew

confluent, the cells were moved to a T75 flask and grown to confluence. This

cell population was a mixed population stable cell line where every cell

expressed the new proteins at a different level.

Dilution cloning and generation of a stable clonal cell line

When it was desired to generate a stable clonal cell line, the mixed population

cell line was grown to confluence and the cells were detached from the

bottom of the flask and centrifuged as described above (see Passaging cells).

The cell pellet was resuspended in 20 mL growth media, and 20 µL were

transferred into another 20 mL of growth medium. From that cell suspension,

100 µL and 120 µL were added to 20 mL of selection media, generating final

200,000- and 167,000-fold dilutions. 200 µL of each dilution was transferred

into 96 wells of a 96-well plate (i.e. one plate per dilution) with the aim to

place only one cell into each well, thus isolating a single cell that can be grown

into a clonal cell line. Both dilutions used here resulted in 0-3 cells per well.

After 48 hours, both 96-well plates were examined and the number of cells

examined 48 hours later. Most of the single cells had grown into small

colonies (= a clone) which were left to grow to about 40-50 % confluence in

the well. At this stage, the cells tend to grow on top of each other rather than

in a defined monolayer which means that 100 % confluence is unlikely to be

achieved. The selection medium was then removed from the wells containing

single colonies and the cells were washed by addition and removal of 100 µL

DPBS. Then, 100 µL trypsin was added to each marked well. After 2-3 minutes,

the trypsin was pipetted up and down to loosen the cells off the bottom of

the well. The trypsin and the cells were then added to 1 mL selection media in

a well of a 24-well plate (1:7 dilution by surface area difference). This was

done for all wells that were marked to contain a single cell colony. Once

confluent in the 24-well plate, the cells were moved to 6-well plates (1:5

dilution) and then to T25 flasks (1:3 dilution) and finally to T75 flasks (1:3

dilution). From there each clone was tested for the presence and functionality

of the transfected gene of interest.

Transient transfection of cells

CHO-K1 cells were seeded into assay plates/flasks and grown to 60-70 %

confluence. Transient transfections were then carried out in the same manner

as described for transfections to generate stable cell lines (see above), but the

volumes of the reagents were adjusted as outlined in Table 2.1. Following

transfection and incubation of cells in a cell culture incubator at 37 °C

overnight, the cells were not exposed a selection medium, resulting in a cell

have taken up the DNA construct will only express the proteins encoded in

the DNA construct transiently (and each cell will do so at a different level), i.e.

do not incorporate the DNA into their nuclear DNA. This is mainly due to time

limitations as cells that are transiently transfected, are used for

Table 2.1 Volumes of reagents used to transiently transfect CHO-K1 cells seeded into 8-well plates, 6-well plates and T75 flasks. The amount of DNA used is total amount for the entire 8-well and 6-well plate assuming transfection of cells in all 8 and 6 wells, respectively. The total volume is the combined volume of solution A and B.

8-well plate 6-well plate T75 flask Solution A Total DNA 1.2 µg 4.5 µg 10 µg OPTIMEM medium 200 µL 300 µL 300 µL Solution B Lipofectamine 20 µL 50 µL 100 µL OPTIMEM medium 260 µL 850 µL 600 µL Total volume 480 µL 1.2 mL 1 mL

Addition per well/flask 60 µL 0.2 mL 1 mL