PMC

Expression of NBS1 and VHL genes loaded into the HAC in CHO cells. (A) FISH analysis of the alphoid^tetO-HAC/NBS1 in CHO cells with specific probes for the BAC vector (in red) and for gene sequences (green). (B) FISH analysis of alphoid^tetO-HAC/VHL in CHO cells. (C) Immunocytochemical staining of CHO cells and (D) isogenic cells with the alphoid^tetO-HAC/NBS1 using Abs against pNBS1 (red) and GFP (green). (E) Western blot analysis of a CHO clone containing alphoid^tetO-HAC/NBS1 with human-specific Abs against pNBS1. NBS1 inserted into the alphoid^tetO-HAC produces a protein of the predicted size.

pVHL expression in VHL-deficient cells. (A) FISH analysis of the alphoid^tetO-HAC/VHL in VHL-deficient 786-0 RCC4 cells. (B) Effect of pVHL on the level of HIF2-α, CyclinD1, and Cdk1/CDC2 proteins. Immunoblots of whole cell extracts isolated from the 786-0 cell line, the same cell line containing alphoid^tetO-HAC/VHL before and after induction of HAC loss. CA9 and PGK1 proteins, which are not affected by pVHL status in 786-0 cells, were used as controls. (C) The level of invasiveness of VHL-deficient 786-0 cells and the same cell line containing alphoid^tetO-HAC/VHL in the presence of FBS. (D) pVHL regulation of HGF-mediated (HGF+) 786-0 cell branching morphogenesis in the absence and presence of functional pVHL (with or without alphoid^tetO-HAC/VHL). (E) pVHL regulation of 786-0 cell migration. pVHL expression in 786-0 cells results in reduction of cellular migration. A clear difference was observed after 7 h when representative fields were photographed. Each experiment was performed in triplicate.

A scheme of consecutive experimental steps from selective gene isolation to its expression in gene-deficient human cells. (A) Direct TAR isolation of the NBS1 and VHL genes from human genomic DNA. TAR vectors contain two gene targeting hooks (yellow and blue boxes). (B) Retrofitting of the circular YAC containing the full-length gene by the pJBRV1 vector containing a 3′ HPRT-loxP-eGFP cassette. Recombination of the BamHI-linearized pJBRV1 vector with a YAC in yeast leads to replacement of the ColE1 origin of replication by the F′ factor origin of replication, allowing subsequent propagation in BAC form. (C) Gene loading into a unique loxP site of the alphoid^tetO-HAC by Cre-loxP recombination in CHO cells. (D) MMCT of alphoid^tetO-HAC/NBS1 or VHL from CHO into gene-deficient cells for complementation analysis (E) Elimination of the alphoid^tetO-HAC/NBS1 or VHL from cells by expression of the tTS fusion construct. (F and G) Analysis of TAR clones containing the full-length NBS1 gene for the presence of exons before (F) and after (G) retrofitting. (H and I) Analysis of TAR clones containing the full-length VHL gene for the presence of exons before (H) and after (I) retrofitting.

pNBS1 expression in NBS1-deficient cells. (A) FISH analysis of the alphoid^tetO-HAC/NBS1 in GM07166 cells with specific probes for the BAC vector (red) and NBS1 gene sequences (green). (B) Western blot analysis of NBS1-deficient GM07166 cells, alphoid^tetO-HAC/NBS1-containing GM07166 cells before and after HAC elimination from the cells by expression of the tTS fusion construct. (C and D) Kinetics of disassembly of γ-H2A.X foci in NBS1-deficient GM07166 cells and the same cells with alphoid^tetO-HAC/NBS1. (C) Immunostaining of irradiated cells. Cells were stained with anti–γ-H2A.X antibodies (red) and with DAPI (blue) after exposition to 1 Gy IR and then collected after the times indicated. (D) Quantitative analysis of disassembly of γ-H2A.X foci. Cells were collected after the time indicated, and the average number of γ-H2A.X foci was quantified over time. (E) Western analysis of GM07166 cells and cells with alphoid^tetO-HAC/NBS1. As predicted, p53, ATM, and KAP1 are phosphorylated in response to irradiation. (F–H) Analysis of colocalization of γ-H2A.X, MRE11, 53BP1, and NBS1 proteins after irradiation. NBS1-deficient cells (GM07166) and the same cells with alphoid^tetO-HAC/NBS1 before and after HAC loss were fixed 2 h after irradiation with 3 Gy and were double-stained with anti–γ-H2A.X, anti-hMRE11, anti-53BP, and anti-hNBS1 antibodies. (F) NBS1 is colocalized with γ-H2A.X in cells carrying the alphoid^tetO-HAC/NBS1. (G) γ-H2A.X and MRE11 are colocalized in foci in a pNBS1-dependent manner. (H) 53BP1 foci formation is not affected by absence of pNBS1.

Immuno-FISH and ChIP analyses of the alphoid^tetO-HAC/NBS1. (A) ChIP analysis of centromeric chromatin in alphoid^tetO-HAC/NBS1 clones 3 and 6. Normal mouse IgG (Top), antibodies against CENP-A, H3K4me2, H3K4me3, and H3K9me3 were used for analysis. The assemblies of these proteins on the alphoid DNA of the original alphoid^tetO-HAC in AB2.2.18.21 cell line (Left), the alphoid^tetO-HAC carrying the human NBS1 gene in NBS1 no. 3 and NBS1 no. 6 cell lines (Right), are shown. The bars show the percentage recovery of the various target DNA loci by immunoprecipitation with each antibody to input DNA. Error bars indicate SD (n = 2 or 3). Analyzed loci are alphoid^tetO (alphoid DNA with tetO motif on the alphoid^tetO-HAC), bsr (the marker gene in the HAC vector sequence), and 3′ and 5′ ends of NBS1. rDNA (5S ribosomal DNA), alphoid^chr.21 (centromeric alphoid DNA of chromosome 21), and sat2 (pericentromeric satellite 2) were used as controls. Immunoprecipitated DNAs were quantified by real-time PCR. (B) Positions of probes for ChIP analysis in the original HAC and in the HAC carrying the NBS1 gene are shown by colored boxes. (C) Immuno-FISH analysis of metaphase chromosome spreads containing the alphoid^tetO-HACs. Cells with the original alphoid^tetO-HAC (AB2.2.18.21) and with alphoid^tetO-HAC carrying the NBS1 gene (clones 3 and 6) were used for analysis. Immunolocalization of the centromeric protein CENP-A on metaphases was performed by indirect immunofluorescence with anti–CENP-A antibody and Alexa 488-conjugated secondary antibody (green). HAC-specific DNA sequence (BAC) was used as a FISH probe to detect the HAC (red). CENP-A and BAC signals on the HACs overlap one another.