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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 60C 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: 94C 1
min, 94C 30 sec / 68C 30 sec-2 min for 15 times with -0.5C per cycle, 94C 30 sec /
60C 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 4C
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: 60C 10 min for reverse transcription, 95C 5 min, 45 cycles of
95C 10 sec / 60C 30 sec / 68C 30 sec, 95C 1 min, 55C 1 min, 80 cycles of 55C 10
sec with +0.5C 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
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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.
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