T ran sfectio n s

The expression plasmid pME-PK7 (kindly supplied by D r M Abe, National Institute of Radiological Sciences, Chiba, Japan) containing the entire coding sequence of murine DNA-PKcs was transformed into Epicurian Coli® SURE 2 Supercompetent cells (Stratagene) following the instructions supplied by the manufacturer (Fukumura, et al.,

1998). Five clones of transformed bacterial cells were picked and cultured in 5 ml LB broth supplemented with 50 pig ml^ ampicillin overnight at 37 °C while shaking and then scaled up to 100 ml cultures that were incubated overnight at 37 “C in a shaker incubator. The cultures were then maxiprepped using the EndoFree Plasmid Maxi Protocol (Qiagen) and the recovered plasmid eluted in 100 pi\ endotoxin-free TE (Qiagen). The

concentration o f plasmid DNA was subsequently measured using an UV- spectrophotometer.

To ensure that the maxiprepped plasmids had not undergone rearrangements or deletions a series of diagnostic digests were performed on each maxiprepped plasmid using the restriction enzymes Apal, BamHl, BgB, EcoRl, Hindill, Pvull, S a d and Xbal (all 10 U

pi\ ^ and manufactured by Promega). Each digest was prepared and carried out as follows. 1 pig of plasmid was mixed with 3 pi\ 10 x buffer (Promega), 0.3 pi\ 10 pig piV^ BSA (Promega), 1.5 pi\ 10 U piV^ restriction enzyme, and made up to 28.5 pi\ with HgO. The digest was then incubated for 3 hours at 37 “C. After 90 minutes an additional 1.5 pi\ 10 U piV^ restriction enzyme was added to the digest. Following completion of the digests the samples were run on an ethidium bromide stained 0.8% (w/v) agarose gel with 1 kb

DNA Ladder Plus (Life Technologies). The different maxiprepped plasmids that displayed the expected digests were then combined into one sample that was subsequently used for transfection.

The DNA-PKcs expressing pME-PK7 and control pp-galCMU plasmids were transfected into immortalized DNA-PKcs -/- MEFs using the Fugene (Roche) method following the reconunendations supplied by the manufacturer. Because a large amount of transfected cells were required for northern and western analysis, each transfection was carried out as

follows. Ten 25 cm^ flasks were seeded with 3.5 x 10^ immortalised DNA-PKcs -/- MEFs each and incubated at 37 “C, 5% CO2 overnight. The following day the 10 flasks were transfected using the Fugene protocol and incubated at 37 “C, 5% CO2 overnight. The next day the 10 flasks were trypsinised and the cells combined and plated into six

150 cm^ flasks that were incubated at 37 °C, 5% CO2 overnight. Each large-scale

transfection was thus an average of 10 separate small-scale transfections. The following day 4 flasks from each transfection were used to isolate total RNA whereas the 2

remaining flasks were used to prepare cell extract for protein analysis purposes. A 25 cm^ flask was co transfected with pME-PK7 and pp-galCMU using the same Fugene protocol. This transfection used half the amount of both plasmids compared to when they were transfected alone. After 48 hours an X-gal assay was carried out to investigate the efficiency of the transfection. The culture media was first aspirated off the cells followed by a brief wash in PBS that was then removed by aspiration. The cells were then fixed in a 1:1 acetoneimethyl alcohol solution for 5 minutes. The fixing solution was then

aspirated off. The cells were then washed three times in PBS, each time the PBS was removed by aspiration. 1.5 ml staining solution (prepared by mixing 2.7 ml PBS, 150 pi\

100 mM KgFeCCN)^, 150 pi\ 100 mM K^Fc(CN\ • 3 H2O, 3 //I 1 M MgCl2 and 37.5 pi\ 40 mg ml * X-Gal) was then added to the cells followed by incubation at 37 °C, 5% CO2 for 3 hours. The flask was then examined under microscope to evaluate the transfection

efficiency as judged by the presence and number of blue cells.

The cell extracts from the large scale transfections were prepared as previously described (Finnie, et a/., 1995). Briefly, (0.5 - 3) x 10^ cells were scraped and pelleted in universal tubes and washed twice in PBS. The cell pellet was then resuspended in 1 ml PBS and transferred to a 1.5 ml eppendorf tube. The cells were then pelleted by centrifuging at 5,(XX) rpm for 5 minutes and the supernatant discarded. Each cell pellet was then snap freezed on dry ice/Industrially Methylated Spirits. 80 jA o f extraction buffer (For 2.5 ml extraction buffer mix 50 //I 1 M Hepes, pH 7.8, 250 pi\0.5 M NaF (Sigma), 225 //I 5 M NaCl, 625 pii Glycerol (Sigma), 1 pi\ 0.5 M EOT A, 1.25 //I 1 M DTT (Sigma), 990.6 pi\

H2O and 357 j A l x Protease Inhibitors Complete Mini EDTA-free (Roche)) was added to each frozen cell pellet. The thawing pellet was then resuspended in the extraction buffer by pipetting. The cells were then lysed by 3 repetitions o f snap freezing for 1 minute, and thawing at 30 “C for 1 minute. The lysed cells were then centrifuged at 14,000 rpm, 4 “C, for 7 minutes. The supernatant containing the proteins was then transferred to fresh tubes in 15-20 //I aliquots and stored at -8 0 “C. The cell debris pellet was also stored at -8 0 “C.

For genotyping the transfection an aliquot of total RNA was removed from each transfection and DNased followed by preparation of first strand cDNA as described in section 2.3. Genotyping of each transfection was subsequently carried out as described using the MQ2, MQ4592 and MQ7 primers (Taccioli, et aL, 1998).

Phosphoimager quantification analysis of LAMA4 transcription was carried out with a Typhoon 8600 variable mode imager (Molecular Dynamics) using Typhoon Scanner Control Version 1 typhoon control software (Molecular Dynamics) and ImageQuant 5.1 image software (M olecular Dynamics).