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Supplementary Materials and methods Construction of targeting vector and screening homologous recombinants λ clones containing UbC genomic DNA including the UbC coding region were isolated by screening λ Fix II 129/SvJ mouse genomic DNA library with probes which correspond to 3’ and 5’ UTR of mouse UbC cDNA and restriction mapped. We designed knockout construct by replacing 2.2 kb UbC coding region with a 1.4 kb fragment containing enhanced green fluorescent protein (GFP) and puromycin cassette for gene disruption and positive selection (Figure 1A). To construct a targeting vector, 1.4 kb fragment from 5’ flanking region and 5.4 kb fragment from 3’ flanking region were inserted with loxP sites and GFP-puromycin (GFP-puro) cassette into pLitmus28 vector containing diphtheria toxin A fragment (DTA) (Yagi et al., 1990). As a result, GFP-puro cassette was flanked by 1.4 kb and 5.4 kb fragments as 5’ and 3’ homologous regions, respectively. Positive selection by puromycin offers advantages because it achieves cell death sooner than more commonly used neomycin and hygromycin (de la Luna and Ortin, 1992). This marker has been successfully used in the selection of other ES cell homologous recombinants (Collins et al., 2000). The use of a promoter trap strategy (e.g., the GFP-puro cassette lacks its own promoter and can only be transcribed if inserted in the correct orientation and proximity to a genomic promoter sequence) further increases the probability of obtaining homologous recombinants (up to 40%, see below). The advantage of DTA is that no additional drug is required for negative selection and thus the pluripotency of the ES cells is better maintained. GFP-puro cassette is flanked by loxP sites so that the selection marker can be excised by Cre recombination. We obtained DR-4 mice, which express four different drug-resistant genes including puromycin, and harvested mouse embryonic fibroblasts (MEFs) for use as feeder cells (Tucker et al., 1997). ES(R1: from F1 progeny of 129/Sv X 129/SvJ) cells (2x107) grown on DR-4 feeders were electroporated with a linearized target construct (12.4 kb) and puromycin-resistant ES cell clones were selected (2 μg/ml for 2 days) and expanded. Total 8 colonies were picked and 7 colonies were expanded. They were screened by PCR using F1 and R1 primers (see below) and 3 clones were confirmed to be homologous recombinants by PCR and Southern blotting (Figure 1B, upper panels). For Southern blot analysis, genomic DNA was prepared from ES cell clones, digested with SacI, and transferred to HybondTM-N membrane (Amersham). Random nonamer-labeled probe with MegaprimeTM DNA labeling system (Amersham) was purified using Sephadex G-50 column to remove unincorporated nucleotides and used for Southern blot analysis. Hybridization was performed in ExpressHybTM solution (Clontech) at 60C for 1 hr. Blot was first hybridized with 5’ probe and then hybridized with DTA probe after stripping. Generation of UbC-/- embryos Two different homologous recombinant ES cell clones were injected into 3.5-day old blastocysts isolated from C57BL/6J females and followed by transfer to pseudopregnant females. Chimeric mice were identified initially for the presence of the agouti coat color and analyzed subsequently for genotype by PCR. Chimeric mice were back-crossed with C57BL/6J mice to identify germ-line chimeras, which were able to generate progeny with agouti phenotype. Germ-line transmission of knockout allele was confirmed by PCR of genomic DNA from tail using primers from 5’ non-coding region (F1) and GFP-puro cassette (R1). After successful germ-line transmission of knockout allele, two lines were established from two separate ES cell clones and maintained in the mixed background of 129/Sv(J) and C57BL/6J. Crossing heterozygous males and females did not result in any UbC-/- progeny, suggesting that UbC-/- mice are embryonic lethal. Genotypes of embryos were confirmed by PCR using primer sets F1/R1 and F1/R2 (Figure 1B, lower panel). F1/R1 amplifies 140 bp fragment from knockout allele, but not from wild-type allele. While, F1/R2 amplifies 100 bp fragment from wild-type allele, but not from knockout allele. PCR cycle profile and the sequence of primers used for genotyping were as follows: 94C 1 min, 94C 30 sec / 68C 30 sec-2 min for 15 times with -0.5C per cycle, 94C 30 sec / 60C 30 sec for 20 times; F1 (5’-TTT ATC GAT GGC TTC TGG TCT GG-3’); R1 (5’GCA CCA CCC CGG TGA ACA GCT C-3’); and R2 (5’-GCA ATC ATA TGC AAA TCA GTG TGA CC-3’). Congenic UbC knockout lines with floxed sequence removed were generated by crossing heterozygous mice with Zp3-Cre mice and subsequently back-crossing to C57BL/6J mice at least 9 times. Zp3 promoter drives expression of Cre specifically in the growing oocytes prior to the completion of the first meiotic division, therefore deleting a floxed sequence specifically in the female germ line (Lewandoski et al., 1997). All mice were kept in plastic cages with ad libitum access to food and water, with 12 hr light cycle (07:00-19:00h). All procedures followed the NIH guidelines with the approval of Stanford University Administrative Panel on Laboratory Animal Care (APLAC). Generation of mice carrying HA-Ub A construct containing the UbC promoter (Schorpp et al., 1996) (a gift from P. Angel, Deutsches Krebsforschungszentrum Heidelberg), six copies of hemagglutinin tagged Ub moieties (Treier et al., 1994) (a gift from D. Bohmann, University of Rochester Medical Center), and a rabbit β-globin intron and poly A excised from the pC4-Fv1E vector (kindly provided by ARIAD Pharmaceuticals) was assembled in an engineered multiple cloning site of pBSKII (Invitrogen). Subsequently, the HA-Ub construct was released from the pBSKII backbone and inserted into the AscI site of the Hprt targeting vector, pMP10 a modification of the described pMP8SKB (Bronson et al., 1996) (a gift from O. Smithies, University of North Carolina at Chapel Hill). HM-1 ES cells (Selfridge et al., 1992) (a gift from D. Melton, The University of Edinburgh) were grown on mytomycin C treated mouse embryonic fibroblasts (MEFs) in high-glucose DMEM supplemented with 15% FBS, L-glutamine, MEM amino acids, sodium pyruvate, penicillin/streptomycin, 0.1 mM β-mercaptoethanol, and leukemia inhibitory factor (LIF). The HA-Ub-Hprt targeting construct was linearized with SwaI prior to electroporation. Targeted HM-1 ES cells were selected on 100 μM hypoxanthine/0.4 μM aminopterin/16 μM thymidine (HAT, Sigma)-supplemented medium starting two days after transfection according to manufacturers protocol. HATresistant colonies were picked after 10 days of selection and screened for homologous recombination by Southern blotting using a probe to the mouse Hprt locus (Bronson et al., 1996). Prior to blastocyst injection ES cells were cultured in HAT-free medium after transition through an intermediate passage in hypoxanthine/thymidine (HT, Sigma)- supplemented medium. Two independent ES cell clones carrying the reconstituted Hprt locus with the engineered HA-Ub gene were injected into C57BL/6J-derived blastocysts and implanted into pseudopregnant recipient mice. Resulting chimeric male mice were bred to C57BL/6J females for germ-line transmission. The mice were housed according to the institutional guidelines. Genotypes of HA-Ub mice were confirmed by PCR using genomic DNA prepared from mouse tails with Hprt wild-type primer set (WT-F: 5’-CTG TCT ACA TAT ATC TCT CC-3’; WT-R: 5’-CAG TTC CAT GCT GGC CCA TCT AC-3’) or the HA-Ub knock-in primer set (HA-F: 5’-CGG AAG GAT CAG GAA CGC TC-3’; HA-R: 5’-GTA GAT GGC AGG AGA TTT GTA A-3’) (Figure 2B, right panel). Histology Embryos were isolated and fixed in 4% paraformaldehyde or Bouin’s fixative for overnight at RT, washed with 70% ethanol, dehydrated, and embedded in paraffin. Four μm sagittal sections were prepared using a microtome, deparaffinized, rehydrated, and stained with hematoxylin and eosin using standard histological procedures. Culture of MEFs Mouse embryonic fibroblasts (MEFs) were generated from 12.5-13.5 days postcoitum mouse embryos. Embryos were harvested, the brain, limbs, and the internal organs were removed, and the carcasses were rinsed with Dulbecco’s Modified Eagle Medium (DMEM) and minced with sterile razor blades. Minced carcasses were resuspended with 1 ml 0.05% trypsin/EDTA (Invitrogen) and incubated at 37°C for 15 min with shaking at 200 rpm. Cell suspension was removed and trypsinization was quenched by adding the equal volume of cell culture media (DMEM supplemented with 10% fetal bovine serum, 20 mM glutamine, and penicillin/streptomycin). The remaining carcasses were trypsinized at least 4 more times. Cell suspensions were centrifuged and resuspended with cell culture media and plated out to 100 mm dish (per embryo) and cultured at 37°C with 95% air and 5% CO2. When the cells are confluent, entire cells were splitted to four to five 100 mm dishes and cultured until confluent. A frozen stock in 5% dimethyl sulfoxide and 95% fetal bovine serum was made from each confluent 100 mm dish. When cells were thawed, the passage number was considered as 2. Early passage MEFs (P<6) was used for the experiment unless otherwise noted. Immunofluorescence and senescence assay For immunofluorescence, MEFs grown on coverslips were fixed in 4% paraformaldehyde containing 10 μg/ml bisbenzamide for 10 min at RT, permeabilized with 0.2% Triton X-100/PBS, and blocked with 0.5% BSA/PBS for 1 hr at RT. Fixed cells were incubated with anti-Ki-67 polyclonal antibody (BD Pharmingen) at 4C overnight, washed with PBS, and incubated with Alexa Fluor 594-conjugated goat antirabbit IgG (Invitrogen) for 1 hr at RT. Then, cells were mounted with Fluoromount G (Southern Biotechnology Associates) and visualized with Zeiss Axiovert 200M microscope with a 20x (numeric aperture=0.50) dry lens. Senescence assay for MEFs was performed using senescence associated (SA) βgalactosidase staining kit (Cell Signaling). Immunoblot analysis and indirect competitive ELISA MEFs treated with 10 μg/ml ALLN or vehicle (DMSO) for 24 hrs were harvested by trypsinization and pellet was resuspended (1x106 cells/100 μl) in hypotonic buffer (10 mM sodium phosphate, pH7.4, with protease inhibitor cocktail from Roche) and incubated on ice for 20-30 min followed by a brief sonication. Total cell lysates were centrifuged at 13000 rpm for 10 min at 4°C and the supernatant was removed to measure protein concentration by BCA protein assay (Pierce). For immunoblot analysis, total cell lysates (50 μg) were subjected to SDS-PAGE followed by immunoblot detection with monoclonal anti-Ub antibody (MAB1510; Chemicon). To prepare samples for ELISA, total cell lysates (24 μg) were treated with 2.4 μg Usp2-cc in the presence of 1.4 mM βmercaptoethanol and 140 mM NaCl in 20 μl reaction volume for 30 min at 37°C. Usp2cc treated total cell lysates were further diluted with 1% BSA/PBS and total Ub levels were measured by ELISA as described previously (Ryu et al., 2006). To measure HA-Ub levels, microtiter plate was coated with 100 ng HA-Ub (Boston Biochem) instead of Ub. After blocking with 1% BSA/PBS, 1000-1 ng HA-Ub standards or cell lysates were added with anti-HA antibody and indirect competitive ELISA was carried out as described (Ryu et al., 2006). Quantitative real-time RT-PCR Total RNA was isolated from MEFs using RNeasy kit (Qiagen) with DNaseI treatment. RNA concentration was determined and 10 ng of total RNA was used as a template. For real-time RT-PCR, we used iScript one-step RT-PCR with SYBR green kit (BioRad) and iCycler system with iCycler iQ software version 3.1 (BioRad), with the following cycle profile: 60C 10 min for reverse transcription, 95C 5 min, 45 cycles of 95C 10 sec / 60C 30 sec / 68C 30 sec, 95C 1 min, 55C 1 min, 80 cycles of 55C 10 sec with +0.5C per cycle for melt curve analysis. The mRNA expression levels of UbC, UbB, UbA52, and UbA80 were normalized to the level of 18S rRNA (Bas et al., 2004). Primers used for real-time RT-PCR were as follows: UbC-F (5’-GTT ACC ACC AAG AAG GTC-3’); UbC-R (5’-GGG AAT GCA AGA ACT TTA TTC-3’); UbB-F (5’-TCT GAG GGG TGG CTA TTA A-3’); UbB-R (5’-TGC TTA CCA TGC AAC AAA AC-3’); UbA52-F (5’-GTC AGC TTG CCC AGA AGT AC-3’); UbA52-R (5’-ACT TCT TCT TGC GGC AGT TG-3’); UbA80-F (5’-TGG CAA AAT TAG CCG ACT TCG-3’); UbA80-R (5’-AAC ACT TGC CAC AGT AAT GCC-3’); 18S rRNA-F (5’-CGG CTA CCA CAT CCA AGG AA-3’); 18S rRNA-R (5’-GCT GGA ATT ACC GCG GCT-3’). Control plasmid DNA was generated by subcloning each cDNA fragment into pCR2.1 vector (Invitrogen) and 108 to 103 copies of plasmid DNA was used as a standard. Statistical analysis Two-tailed unpaired Student’s t-tests were used to compare the data between two groups. p < 0.05 was considered to be statistically significant. Supplementary References Bas, A., Forsberg, G., Hammarstrom, S. and Hammarstrom, M.L. (2004) Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphatedehydrogenase for normalization in real-time quantitative reverse transcriptasepolymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol, 59, 566-573. Bronson, S.K., Plaehn, E.G., Kluckman, K.D., Hagaman, J.R., Maeda, N. and Smithies, O. (1996) Single-copy transgenic mice with chosen-site integration. Proc Natl Acad Sci U S A, 93, 9067-9072. Collins, E.C., Pannell, R., Simpson, E.M., Forster, A. and Rabbitts, T.H. 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