Mutation assay

2.4.1 General mutation assay

As any o th er m u tatio n assay, th is assay can be divided into th ree separate parts:

(a) The first p a rt involves the exposure of cells to a potential m utagen or well defined m utagen e.g X-rays.

(b) The second p a rt is defined a s the expression period. This is th e tim e w hich is required for the genomic dam age to effectively alter the gene p ro d u ct w hich is then assayed for to determ ine th e induced m utation frequency.

(c) The final p a rt involves plating out of the m utagenised population of cells in a selection m edium w hich norm ally co n tain s a toxic-drug analogue of the norm al gene product. A sim ultaneous viability assay is also carried o u t at this point to allow the expression of m utation as the induced m u ta tio n frequency per survivor. T hese ste p s have been sum m arised in figure 2.1.

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M utagen

Expression period (4 days)

Trypsinise

M utation assay Viabilty assay

TK- mutant

colory Viable colony

Cells seeded in TFT (Sitg/ml)

supplem ented m edium Cells seeded in norm al m edium (MEM)

12 days INCUBATE AT 37^C 8 days

Fix, stain and count colonies

F igure 2.1. S um m ary of th e step s involved in th e isolation of tk~

m u ta n t cells following tre a tm e n t of h a m ste r cells w ith a specified m utagen.

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D u rin g th e co u rse of th e project, different tre a tm e n ts w ith th e m u tag en s w ere carried out hence each treatm en t is described in the resp ec tiv e c h a p te rs u n le s s o th erw ise m en tio n ed . H ow ever, th e p ro ced u re following th e tre a tm e n t w as n early id en tical in all th e experim ents hence is described below.

Following th e exposure of th e cells to th e m utagen, th e cells w ere seeded in 75 cm2 flasks (Sterlin) an d in cu b ated a t 37°C for 4 days expression tim e. This tim e w as found to rep resen t th e optim um tim e for th e recovery of tk- m u ta n t cells. D uring th e co u rse of th e expression period, cells were m aintained in th e exponential p h ase of grow th by p assag in g confluent cell c u ltu res. A fter th e ex p ressio n period, cells w ere trypsinised an d plated o u t a t a co n cen tratio n of lO^-lO® cells p er d ish (10 cm, Sterlin) w ith 10 m l of MEM/TFT, the TFT diluted down from the stock to the w orking co n cen tratio n of 3 p g /m l. This concentration w as found to m axim ize th e recovery of the

tk- m u ta n t cells. D ishes were incu b ated for 12 days in a n hum idified In cu b ato r a t 37°C w ith 5% CO2 in air. After th is period, colonies were fixed an d stain ed w ith G iem sa s ta in (BDH Ltd) before co u n tin g th e n u m b er of TFT^ colonies.

2 .4 .2 Calculation o f induced m utation frequencies

M utation frequencies in the control cell populations were calculated as follows:

M

' ' o

W here Nq is the n u m b er of spontaneously induced m u ta n t colonies in

th e control cell population, Vo is th e n u m b er of viable cells in th e sam e u n irrad iated population a t th e tim e of m u ta n t selection, Mq is

th e m u ta tio n frequency p er viable cell (or p e r survivor) in th e u n irrad iated population.

Sim ilarly, the m u tatio n frequency in th e treated cell populations w ere calculated as follows:

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W here, Nx is th e n u m b er of m u ta n t colonies in th e trea te d cell p op u latio n , Vx is the n u m b er of viable cells in th e sam e treated cell population determ ined a t the tim e of m u ta n t selection, an d Mx is the in d u ced m u tatio n frequency p er viable cell (or p er survivor) in th e treated cell population.

Hence the n et induced m utation frequency is given by:

Mx - Mo

2.4.3 Viability assay

After th e 4 days expression period, trypisinsed cell su sp en sio n s w ere d ilu te d dow n in M EM /FC S to give 100-200 cells p er 5cm d ish supplem ented w ith 5 ml M EM /FCS. D ishes were incubated for 8 days in a n hum idified in cu b ato r a t 37°C w ith 5% CO2 in air before fixing an d counting the n um ber of viable colonies.