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A chromosome engineering strategy that allows the generation of chromosomes with this …
Biology Articles » Bioengineering » Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing » MATERIALS AND METHODS
Cells were exposed to 10 ng/ml of colcemid (Sigma) for1 h. Trypsinized cells were centrifuged at 1000 rpm(300 g) for 10 min in a swinging bucket rotor. Thecell pellet was resuspended in 75 mM potassium chloridefor 15 min at 37°C, recentrifuged at 1000 rpmfor 10 min and fixed in 3 : 1 methanol : aceticacid. Fixed cells were added drop-wise to microscope slidesto make mitotic chromosome spreads using standard methods (27).
Fluorescence in situ hybridization
Slides with mitotic spreads were baked at 85°C for 20 minand then treated with 0.1 mg/ml RNase for 1 h at 37°C.After RNase treatment, the slides were washed in 2x SSC (1xSSC is 150 mM NaCl and 15 mM sodium citrate) withthree changes for 3 min each and dehydrated in 70, 90 and100% ethanol for 3 min each. The chromosomes were denaturedin 70% formamide in 2x SSC at 70°C for 3 min and WCPswere used according to the manufacturer's recommendations (AmericanLaboratory Technologies and Vysis). Detection of digoxigenin-dUTPprobes used a three-step incubation of slides with sheep FITC-conjugatedanti-digoxigenin antibodies (Roche) followed by rabbit FITC-conjugatedanti-sheep antibodies (Roche) followed by goat FITC-conjugatedanti-rabbit antibodies (Jackson Laboratories). Slides were stainedwith DAPI (12.5 µg/ml) or propidium iodide (0.3 µg/ml),cover slipped and viewed under UV fluorescence with FITC filters(Zeiss).
Mitotic chromosome spreads were prepared as described in theprevious section. Slides were treated with RNase at 100 µg/mlfor 1 h at 37°C and washed in 2x SSC and dehydratedin 70, 90 and 100% ethanol. Chromosomes were denatured at 75°Cfor 3 min in 70% formamaide/2x SSC, followed by dehydrationin ice cold 70, 90 and 100% ethanol. Probe cocktails (Vysis)were denatured at 75°C for 10 min and pre-hybridizedat 37°C for 30 min. Probes were applied to slides andincubated overnight at 37°C. Post-hybridization washes consistedof three 3-min rinses in 50% formamide/2x SSC, three 3-min rinsesin 2x SSC and finally three 3-min rinses in PN buffer (0.1 MNa2HPO4+0.0 M NaH2PO4, pH 8.0, +2.5% Nonidet NP-40), allat 45°C. Slides were then counterstained with either propidiumiodide (2.5 µg/ml) or DAPI (15 µg/ml)and viewed under UV fluorescence (Zeiss).
Replication timing and immunofluorescence
The BrdU replication timing assay was performed as previouslydescribed (8). Asynchronously growing R175B cells were exposedto a pulse of 20 µg/ml of BrdU (Sigma) for 15 min,washed with PBS and chased in media containing 0.2 mM thymidine.Mitotic cells were harvested in the absence of colcemid. Thecells were treated with 75 mM KCl for 15 min at 37°C,fixed in 3 : 1 methanol : acetic acid anddropped on wet slides. The chromosomes were denatured in 70%formamide in 2x SSC (1x SSC is 150 mM NaCl and 15 mMsodium citrate) at 70°C for 3 min. Incorporated BrdUwas detected using a FITC-labeled anti-BrdU antibody (BectonDickinson). Slides were stained with propidium iodide (0.3 µg/ml),cover slipped and viewed under UV fluorescence (Zeiss).
Three micrograms of the 5'-AP-Neo plasmid was linearized ata unique NotI site, electroporated (300 V, 950 µFin PBS; Bio-Rad) into HTD114 cells and grown under 500 µg/mlG418 (Geneticin, Gibco) selection for 10–14 days. G418-resistant(NeoR) colonies were then pooled and expanded. Three microgramsof the Hyg-3'RT plasmid was linearized at a unique NotI site,electroporated (300 V, 950 µF in PBS; Bio-Rad)into the pooled NeoR cells and grown under 500 µg/mlG418 and 200 µg/ml Hygromycin B selection for 10–14days. One hundred and ten individual NeoR, HygR colonies (‘P-clones’)were picked, expanded and subsequently transiently co-transfected(Lipofectamine, Gibco) with 1 µg of a green fluorescentprotein (GFP) expression plasmid (pCSGFP) and either 3 µgof a Cre recombinase expression plasmid (pBS185, Gibco) or anempty vector control (pBluescript SK, Stratagene). Average transfectionefficiencies were between 5 and 10%, as determined by GFP expression.Cre-transfected cells were grown for 14–21 days in 10 µg/mlazaserine and 10 µg/ml adenine (AA selection). Theresulting Aprt+ colonies were pooled to generate 83 independentpools and expanded. A preliminary description of this systemwas described previously (9). Alternatively, individual Aprt+clones were picked and expanded to approximately 4x106 cellsand analyzed for the presence of ‘new’ rearrangementsusing FISH with WCPs.
Deletion of neomycin and hygromycin genes
P27 and P175 were co-transfected (Lipofectamine, Gibco) with3 µg of a Cre-expression plasmid (pBS185) and 1 µgof a GFP-expression plasmid (pcsGFP). After 24 h, the cellswere trypsinized, washed once in Hank's balanced salt solution(Gibco) and 1x106 cells were subjected to FACS with gating setto collect GFP-expressing cells. Typical transfection efficienciesbefore FACS, based on the number of GFP-positive cells, werebetween 15 and 25%. After FACS, the collected cell fractionswere suspended in 1 ml of fresh Hank's balanced salt solutionand consisted of approximately 3x105 cells with 85–95%being GFP+. The GFP+ cells were plated in serial dilutions,to allow single colony growth, onto 15-cm tissue culture dishescontaining DMEM supplemented with 10% fetal bovine serum. Individualcolonies were picked, expanded and challenged to grow in mediacontaining 500 µg G418 or 200 µg HygromycinB. Clones that failed to grow in both G418 and Hygromycin Bwere expanded and subjected to southern analysis to confirmloss of the Neo and Hyg markers. The absence of translocationswas confirmed using FISH with appropriate WCPs.
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