Targeting vector construction
A targeting vector was made to produce a deletion in Mrad1. We used the promoterless selection strategy to obtain a high efficiency of homologous gene targeting [20]. The targeting vector was constructed in three steps starting with pBluescript SK(+) vector. First, the 5'end fragment, a 1523 bp Mrad1 sequence between exon 2 and exon 3, was generated by PCR from 129 SvEv mouse genomic DNA with primers:
5'-GTCTCAGGTTTTCACACATCTTCC-3' and 5'-CTACGCGTCGACCTTCCTGAATGACAAATTCCTG-3' (Fig. 1A). The PCR product was cut with Kpn 1 and Sal 1, and subcloned into pBluescript SK(+). Second, the neo gene was amplified from pRc/CMV2 vector without the promoter and ATG using primers:
5'-CTACGCGTCGACATTGAACAAGATGGATTGCACGC-3' and 5'-AAGGAAAAAAGCGGCCGCAGACATGATAAGATACATTGATGAG-3'. Then, the PCR product was cut with Sal 1 and Not 1, and inserted in frame with Mrad1 into the plasmid constructed in the first step. Third, the 3'end fragment, 5591 bp long between intron 3 and intron 6, was generated by PCR from 129 SvEv mouse genomic DNA with primers:
5'-AAGGAAAAAAGCGGCCGCCTACTACAACTACTGCTACTAC-3' and 5'-TCCCCGCGGCACAGGACAGTACAGTAAGTCG-3'. The product was cut with Sal I and Sac II, and inserted into the vector constructed in the second step. This yielded the final targeting construct with the selectable neo gene, which was linearized with Kpn 1 prior to transfection into ES cells.
Growth of ES cells, gene targeting, and generation of Mrad1-deficient cells and mice
ES cells derived from 129 SvEv mice were cultured by established methods [38]. ES cells used to make gene-targeted mice were grown on feeder cells, electroporated with targeting vector linearized by Kpn 1, and then grown in the presence of G418 at 300 μg/ml. The G418-resistant clones were picked, expanded and subjected to Southern blot hybridization and PCR analyses to identify Mrad1+/- targeted clones. Positive clones were injected into C57BL/6 blastocysts. Chimeric offspring were born and mated to C57BL/6 mice to confirm successful germ line transmission of the targeted Mrad1 allele. Genomic DNA from tails was analyzed by Southern blot hybridization and PCR analyses. Mrad1 heterozygous mutant mice were intercrossed and maintained.
Southern blotting and PCR assays to assess genotypes
For Southern blotting, genomic DNA was isolated from ES cells and tails of mice using published methods [22]. DNA was digested with Hind III, separated on a 0.7% agarose gel, then transferred to a nylon membrane, and hybridized to a 32P-labeled probe, which was generated by PCR using primers:
5'-GTGGCCTAGGTGGTTGCGTATCTGAAC-3' and 5'-GTCGGCTCCGAGAAGAAGGATGCTCC-3' in conjunction with mouse genomic DNA as template.
To genotype ES cells and mice by PCR, the reaction was performed using genomic DNA templates and the following primer pair:
5'-GTCTCAGGTTTTCACACATCTTCC-3' and 5'-GCTTATATTCTAGAAACCTTCCTGTATG-3'. PCR conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 3 min, with a final extension at 72°C for 10 min.
Morphological analysis of mouse embryos
Mouse embryos were obtained at several stages of gestation, including E6.5, E7.5, E8.5, E10.5, and E11.5. All dissections were performed in 1 × PBS. Whole embryos were rinsed with 1 × PBS. Pictures of whole embryos were taken while viewed by a Wild Heerbrugg dissecting microscope.
Preparation and in vitro culture of keratinocytes
Full-thickness skin removed from newborn (1-2 days old) mice was treated with 0.25% trypsin overnight at 4°C. The epidermis was peeled off from the dermis and minced into pieces smaller than 1 mm. They were placed into a sterile flask, then dispersed by stirring into single cells for 30-60 min, then suspended in Keratinocyte-SFM medium with supplements (Invitrogen). Cells were first incubated in dishes coated with collagen type I at 34°C in 5% CO2 for 12 h to allow cells to attach to the bottom. Afterwards, unattached cells were removed by washing with PBS. Attached cells were further cultured in fresh medium, which was replaced every 2 days.
Western blotting
For preparing protein from epidermis, full-thickness skin removed from newborn mice was treated with 0.25% trypsin overnight at 4°C. The epidermis was peeled off from the dermis and dispersed in lysis buffer. To prepare cell lysate, keratinocytes incubated for 3 days were either left untreated or treated for 24 h with 0.15 μg/ml DMBA (Sigma). Then, the cell lysate was prepared in 1× SDS-sample buffer, to a final concentration of 104 cells/μL. Fifty μg of protein were resolved on a 10% SDS-PAGE gel, and proteins were transferred to a polyvinylidene difluoride membrane. The membrane was probed consecutively with primary and peroxidase-conjugated secondary antibodies. Primary and secondary antibodies used in this study are mouse anti-GAPDH (KangChen, China), mouse anti-p21 (Santa Cruz), mouse anti-p53 (Oncogene), peroxidase-conjugated anti-rabbit IgG (A9169, Sigma) and peroxidase-conjugated anti-mouse IgG (A9044, Sigma).
DMBA-TPA induced skin tumor formation
Mice (7-8 weeks old) were shaved on their backs 2 days before tumor induction. To induce tumors, the shaved dorsal skin of mice was treated topically with 15 μg of DMBA (Sigma) in 100 μL acetone once. After 1 week, each animal received subsequent topical treatments of 2 μg of TPA (Sigma) in 100 μL acetone twice weekly for 17 weeks. Treated areas were examined weekly for the presence of tumors, which were scored positive if they reached at least 1 mm in diameter.
Histologic analysis and Immunohistochemistry
Dorsal skin samples and tumors were fixed in 4% paraformaldehyde at 4°C overnight, embedded in paraffin, and sectioned as 8-μm slices. The sectioned tissues on slides were stained with H&E [39, 40]. Immunohistochemical staining was carried out using a kit (ImmunoCruz Staining Systems, Beijing Zhongshan Golden Bridge Biotechnology). The endogenous peroxidase activity in the specimens was blocked by treatment with 0.3% H2O2 and samples were then rinsed with PBS. The specimens were probed consecutively with primary antibodies against Keratin 14 (BAbCo), secondary antibody biotin-conjugated goat anti-rabbit IgG, and horseradish peroxidase-streptavidin complex, and then visualized by diaminobenzidine. Afterwards, sections were counterstained with hematoxylin.
Proliferation assay
Keratinocytes were isolated as described above and seeded into 6-well plates (5 × 105 cells per well) containing Keratinocyte-SFM medium with supplements. Cell numbers were determined every 2 days.
Cell cycle analyses
The cell cycle profiles of cells in different phases were determined using previously established methods [41]. Briefely, 1 × 107 keratinocytes were plated in each 10-cm dish. After incubation for 3 days the cells were mock-treated or treated with 0.15 μg/ml DMBA (Sigma) for 24 h, then processed and stained with propidium iodide (PI), and analyzed by a FACSCalibur cytometer (Becton Dickinson). To assess DNA synthesis, 10 μM BrdUrd was added to medium and cells were pulse labeled for 40 min. Cells were then processed, probed with FITC-conjugated anti-BrdUrd antibody (Becton Dickinson) and stained with PI. Flow cytometric analyses were performed on a FACSCalibur.
Apoptosis assay
Keratinocytes incubated for 4 days were mock-treated or treated for 24 h with 0.15 μg/ml DMBA, trypsinized for 10 min using 0.1% trypsin at 37°C, washed twice with cold PBS, then resuspended in 1× binding buffer [10 mmol/L HEPES (pH 7.4), 140 mmol/L NaCl, and 2.5 mmol/L CaCl2] at a concentration of 1 × 106 cells/mL. Then cells were stained with Annexin V-FITC (Jingmei Biotech) and PI for 15 min at room temperature before flow cytometric analysis.
Neutral comet assay
Keratinocytes were cultured in standard medium for 4 days. The comet assay was carried out according to the manufacturer's instructions (Trevigen). Briefly, cells at a concentration of 1 × 105/mL were mixed gently with premelted low-temperature-melting agarose at a volume ratio of 1 to 10 (v/v) and spread on glass slides. The slides were then submerged in precooled neutral lysis buffer at 4°C for 30 min. After rinsing, the slides were equilibrated in Tris-borate EDTA solution, electrophoresed at 1.0 V/cm for 20 min, and then stained with PI. Fluorescence images for at least 50 nuclei were captured using a Nikon microscope and analyzed by CASP-1.2.2 software (University of Wroclaw) for tail moment (i.e., the geometric mean of fluorescence on the tail from the nucleus).
Statistical analysis
All statistical analyses were performed using statistical software package SPSS Version 10.0. The Kaplan-Meier PL method [23] was used for comparison of the relative risks of tumor development induced by DMBA-TPA between the mice with the two different Mrad1 genotypes. We designed the tumor development experiment to meet a set of conditions so the Log-Rank Test in the Kaplan-Meier PL method could be used. The Student's t test was performed to determine statistical significance of the differences for the comet assay. Wilcoxon rank-sum test was used to compare the difference in tumor numbers between the two groups of mice having different Mrad1 genotypes. In all the above analyses, a P value of < 0.05 was considered statistically significant. Skewness was used to compare the difference of tumor size distributions between Mrad1 wild type and heterozygous mice.
Immunofluorescence assay
Keratinocytes grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed in PBS twice, incubated in PBS containing 0.5% Triton-X100 for 15 min and in PBS containing 5% BSA and 0.1% Triton-X100 for 1 hr, and washed in PBS once, followed by incubation with anti-phospho-H2AX (Upstate) primary antibody (1:100 dilution) in PBS containing 5% BSA and 0.1% Triton-X100 for 1 hr at room temperature. Afterwards, the coverslips were washed two times for 5 min each in PBS and incubated with Texas Red -conjugated anti-mouse antibody (1:100 dilution in PBS containing 5% BSA and 0.1% Triton-X100) for 1 hr at room temperature. Finally, the coverslips were counterstained with DAPI (10 ng/ml). The images were captured using a fluorescence microscope.
Quantitative real-time RT-PCR
Total RNA was isolated from mouse tumors (3 wild type and 3 Mrad1 heterozygous tumors) or keratinocytes cultured for 4 days using the RNeasy Mini kit, as described by the manufacturer (QIAGEN). Two μg total RNA were reverse transcribed in a 20 μL reaction volume to form cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR was performed using the StepOnePlus system(ABI) with SYBR Green I (Takara) to label amplified DNA. A standard curve method of quantification was used to calculate the expression of target genes relative to the housekeeping gene β -actin. Experiments were performed thrice. The following primer pairs were used for the PCR reactions: Mrad9, 5'-GCCTCTTACTATCCACTTCG-3' and 5'-AGCCCTCATTGCCTCC-3'; Mrad1, 5'-GCCCTATTTCAGGTTGT-3' and 5'-TGCCCATCTTCATTTCT-3'; Mhus1, 5'-TCCCTGTCTTACCGTGTC-3' and 5'-CTCCCTTTAGGTTTGCTT-3'; β-actin, 5'-GTAAAGACCTCTATGCCAACA-3' and 5'-GGACTCATCGTACTCCTGCT-3'. We used the following PCR procedure: 94°C for 3 min, then 40 cycles of 94°C for 15 s, 55°C for 20 s, 72°C for 19 s, and a final extension at 72°C for 3 min.
