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  • Materials and methods Construction of the Chk


    Materials and methods Construction of the Chk-YB-1b-Neo vector. Approximately 107 plaques from a chicken genomic library established in EMBL3 vector (Clontech Palo Alto, CA) was screened and one clone containing a 12kb insert was selected. Digestion of this clone with SalI and EcoRI restriction enzymes released five different fragments of 4.5, 2.5, 2.1, 1.8, and 0.6kb. Southern hybridizations indicated that the 2.1, 1.8, and 0.6kb fragments carried sequences corresponding to the Chk-YB-1b cDNA. Therefore, these fragments were cloned into the plasmid pGEM-3Z vector and sequenced. The pCINeo plasmid DNA (Promega, Madison, WI) was digested with SspI and BamHI, and the 1.4kb fragment containing the SV40-NeoR (neomycin-resistance) gene was eluted and end-filled with Klenow. pGEM-Chk-YB-1b was digested with StyI, the large fragment was eluted, end-filled with Klenow, and ligated with the 1.4kb SV40-NeoR fragment using T4 DNA ligase. Following transformation of Escherichia coli Top10 F+ competent cells, appropriate clones were selected and DNAs were isolated. Transfection and selection of G418-resistant DT40 cells. DT40 STF 31 [18], [19], obtained from Dr. Koi, National Institute of Environmental Sciences, Durham, NC, were grown at 37°C in a humidified incubator containing 5% carbon dioxide in DMEM (high glucose) containing 10% fetal calf serum (Atlanta Biologicals, Norcross, GA), 10% tryptose phosphate broth, 2% chicken serum (Sigma, St. Louis, MO), penicillin, and streptomycin. Cells were sub-cultured every two days. About 5×105 DT40 cells in mid-log phase of growth were transfected with 5.0μg pGEM3Z-Chk-YB-1b-Neo DNA using 25μl LipofectAMINE (Life Technologies, Rockville, MD) following the protocol suggested by the manufacturer. Eighteen hours after transfection, cells were transferred to the selection medium containing 700μg/ml G418. Selection medium was changed every 48h. After 25 days of selection, the G418-resistant DT40 cells were sub-cloned by diluting cultures to about 1cell/10ml and plating 1ml per each well of two 96-well plates. After 10 days, we found that G418-resistant cells began to grow in 20 different wells, which were numbered as Chk-YB-KO1–20. Southern blots and PCR analysis. Genomic DNA was isolated from wild-type DT40 as well as from different clones of DT40YB-1b+/− using standard procedures [24]. DNA was then digested with different restriction enzymes, separated on an agarose gel, and subjected to a Southern blot with either the Chk-YB-1b or the Neo specific cDNA probes, as described [24]. To confirm the insertion of SV40-Neo fragment into the intended place, PCR was performed using the primers F1 (YB1 gene 1672–1649C) and R1 (pCINeo1984–1960C). The presence of one wild-type allele in DT40YB1+/− cells was confirmed by using primers F2 (nucleotides 653–674 of YB1 cDNA) and R2 (nucleotides 1103–1083C) in a PCR using Perkin–Elmer Gene Amp 2400 thermocycler. Cycling conditions include one cycle at 94°C for 5min followed by 35 cycles, each for 1min at 94°C, 1min at 55°C, and 1min at 72°C, with one final cycle at 72°C for 7min to complete elongation. Tenμl PCR products were separated on 1% agarose gel in TBE buffer. Characterization of the heterozygous DT40YB1cells. Cells were plated at a very low density on day 1 (50,000 cells per 100mm dish, 10ml medium). Everyday, cells were counted using a hemocytometer. Fluorescence-activated cell sorter (FACS) analysis was performed as follows: cells (2×106cells/ml) were fixed with 40% ethanol, washed with phosphate-buffered saline (PBS), and treated with 100μg/ml ribonuclease-A at 37°C for 20min. Fiveμl propidium iodide (0.5mg PI/ml in PBS) was added to each ml of cell suspension, mixed thoroughly, and incubated in dark at 37°C for 30min. Fixed cells were placed on ice and visualized under a fluorescence microscope or subjected to FACS analysis. For fluorescence microscopy, cells were observed under a Zeiss fluorescence microscope at 400× magnification in rhodamine red channel and photographed using a SPOT digital camera. FACS analysis of propidium iodide-stained cells was performed at the Cell and Immunology core facility of the University of Missouri-Columbia. Chromatin condensation was detected by staining with 10μg/ml Hoechst 33258 (Sigma, St. Louis, MO) followed by fluorescence microscopy using a DAPI filter.