Construction of the targeting vector.
The STAM1 genomic locus was isolated from the λFixII mouse 129/Sv genomic library (Stratagene) using a 5′ region of STAM1 cDNA. This targeting construct replaces a 0.6-kb PstI-PstI genomic fragment encompassing exons 3 and 4, flanked by 6.5-kb (XhoI-PstI) and 1.4-kb (PstI-PstI) genomic sequences derived from the 129/Sv genomic library (see Fig. ). A conditional targeting vector was constructed to delete exons 3 and 4 of the STAM1 gene. A HindIII-XbaI-digested 0.5-kb fragment of the STAM1 genomic DNA containing exons 3 and 4 was inserted between the loxP site and the pGK-neo of the vector (ploxpNEO), which bears a pGK-neo gene cassette flanked by a pair of loxP sequences for positive selections and a diphtheria toxin A-chain gene cassette lacking a polyadenylation site for negative selection. The 6.5-kb (XhoI-PstI) genomic fragment 5′ upstream of exon 3 was blunted and ligated into the XhoI site of the ploxpNEO. The 3′ downstream 1.4-kb (PstI-PstI) genomic fragment was blunted and ligated into the EcoRI site of the ploxpNEO (Fig. ).
FIG. 2. Comparison of histological analysis of thymus and thymocyte counts. Size of thymus (A and B), hematoxylin-eosin-stained sections of thymus (C and D), and TUNEL-stained sections of thymus (E and F) from 4-week-old Lck-Cre (+) STAM1flox/+ (more ...)
FIG. 1. Generation of T-cell-specific disruption of both STAM1 and STAM2 genes. (A) Schematic representation of the mSTAM1 cDNA, stam1 genomic locus, targeting vector, and mutated stam1 locus. The positions of stam1 axons are shown as boxes. Restriction sites: (more ...) Generation of “floxed” STAM1 mice.
The linearized targeting vector was electroporated into J1 embryonic stem (ES) cells. Homologous recombination events were assessed by Southern blot hybridization. The targeted STAM1 loxP-flanked (“floxed”) ES clone was then injected into C57BL/6 blastocysts and transferred to foster mothers to obtain chimeric mice. The F1 heterozygous mice carrying the STAM1 floxed mutation were identified by Southern blot hybridization and intercrossed to produce F2 homozygous offspring. The F2 mice were genotyped by Southern blot hybridization and by PCR with DNA from tail biopsy specimens (Fig. ).
Generation of Lck-Cre STAM1flox/flox STAM2−/− mice.
mice were generated by gene targeting (M. Yamada, N. Ishii, K. Murata, H. Sasaki, and K. Sugamura, submitted for publication). STAM1flox/flox
mice were mated with STAM2−/−
mice to generate STAM1flox/+
mice were then intercrossed to generate STAM1flox/flox
mice. Lck-Cre transgenic mice (31
) were then bred with the STAM1flox/flox
mice to generate Lck-Cre(+) STAM1flox/+
mice. These mice were mated with STAM1flox/flox
mice. Offspring carrying Lck-Cre (+) STAM1flox/flox
and Lck-Cre (+) STAM1flox/+
were used for further analysis. These mice were genotyped by PCR with DNA from mouse tail and thymus (Fig. ).
The following oligonucleotide primers were used: primer A, CGGGACCAGAGGAAAAGCACCTGTCAC; primer B, ATCAGTGTACAAATGGGAAGGTATTAT; and primer C, AACCCCCAAAATTTACCAGAGAACTTC. PCR conditions were as follows: denaturation at 94°C for 5 min, followed by 40 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. The wild-type and lox alleles gave rise to PCR-amplified fragments of 325 and 405 bp, respectively.
Reverse transcription-PCRs (RT-PCRs) were carried out with total RNA derived from mice thymocytes or liver as the template. The total RNA was prepared using TRIzol (Gibco-BRL). The first-strand synthesis was performed using the Superscript Preamplification System (Gibco-BRL). PCRs were performed in a 50-μl mixture consisting of 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxynucleoside triphosphate mixture, a 1 μM concentration of each primer, 1.25 U of Taq DNA polymerase (TAKARA SHUZO, Kyoto, Japan), and 2 μl of the RT reaction mixture as a template. PCR conditions were as follows: denaturation at 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 57°C, and 1 min at 72°C. The following oligonucleotide primers were used: STAMex1F (primer A), CCCTTCGACCAGGATGTTGAGAAAGCA; STAMex5R (primer B), CCCTTCGACCAGGATGTTGAGAAAGCA.
Immunoprecipitation and immunoblotting.
Immunoprecipitation and immunoblotting were performed according to a modification of a previously described method (20
). Briefly, thymocytes from mice were lysed in NP-40 lysis buffer (1% Nonidet P-40, 25 mM Tris-HCl [pH 7.5], 140 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, aprotinin [20 μg/ml], 1 mM Na3
). The supernatants of the lysates were subjected to immunoblotting, or immunoprecipitated with a monoclonal antibody (MAb) specific for STAM1 (33
). The lysates or immunoprecipitates were separated by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20, the filters were incubated with the primary antibodies (Abs), followed by incubation with anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG coupled with horseradish peroxidase, and visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The Abs for extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, Jun N-terminal protein kinase 1/2 (JNK1/2), p-JNK1/2, p38 mitogen-activated protein kinase (p38MAPK), phospho-p38MAPK (p-p38MAPK), p-STAT5, and p-Akt were purchased from Cell Signaling Technology. The Abs for p-Tyr and Akt were purchased from Upstate Biotechnology. The Ab for STAT5a was purchased from Santa Cruz Biotechnology.
Histological analyses of thymus.
Mice were perfused with PBS followed by 4% paraformaldehyde-PBS. The thymuses were removed for processing, embedded in paraffin, and then sliced into 10-μm-thick sections with a microtome. For histological analyses, the sections were stained with hematoxylin-eosin. For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, fresh frozen thymuses of mice were sliced into 10-μm-thick sections with a cryostat. Terminal transferase labeling of fragmented DNA in the sections was then performed with a TACS 2 TdT Kit, HRP-Blue Label (TREVIGEN) according to the manufacturer's protocol.
Thymocytes were suspended in PBS supplemented with 3% fetal bovine serum (FBS). They were preincubated in normal mouse serum to prevent the nonspecific binding of labeled MAbs to the cell surface. They were then stained with MAbs conjugated with fluorescein isothiocyanate, phycoerythrin, or biotin for 30 min at 4°C. The cells were washed with PBS-3% FBS, and the biotinylated Abs were developed with streptavidin-allophycocyanin (PharMingen). All the MAbs were purchased from PharMingen. The surface stainings with MAbs were analyzed with a FACSCalibur flow cytometer (Becton Dickinson) in two- or three-color mode using CellQuest software.
Preparation of thymocyte subpopulations.
To prepare double-negative (DN), double-positive (DP), CD4-single-positive (CD4 SP), and CD8-single-positive (CD8 SP) thymocytes, we used the magnetic cell sorting system with autoMACS (Miltenyi Biotec). Briefly, total thymocytes were labeled with CD8 microbeads and separated by autoMACS. CD8-negative fractions were labeled with CD4 microbeads and separated. Negative fractions were enriched in DN thymocytes, and positive fractions were enriched in CD4 SP thymocytes. CD8-positive fractions were also labeled with CD4 microbeads and separated. Positive fractions were enriched in DP thymocytes and negative fractions were enriched in CD8 SP thymocytes. The purity of each thymocyte subpopulation was confirmed by flow-cytometric analysis and was 90 to 95%.
Single-cell suspensions of thymocytes in RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-mercaptomethanol, penicillin, and streptomycin were plated in 96-well plates at a density of 2 × 105 cells (total thymocytes) or 5 × 104 cells (CD4 SP thymocytes) per well in 100 μl of medium. Stimuli were added as indicated, and cells were cultured for 42 h. The stimuli were recombinant murine IL-2 (20 ng/ml; PeproTech), recombinant murine IL-7 (10 ng/ml; PeproTech), plate-coated anti-CD3 MAb (3 μg/ml; PharMingen), phorbol myristate acetate (10 ng/ml; Sigma), and ionomycin (1 μg/ml; Sigma). The cells were then pulsed with [3H]thymidine and harvested after 6 h. The incorporated [3H]thymidine was counted with a MicroBeta liquid scintillation counter (Amersham Pharmacia Biotech).
Cell survival assay.
To assess the spontaneous death of DP or CD4 SP thymocyte subsets, cells were cultured for 12, 24, or 48 h in RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-mercaptomethanol, penicillin, and streptomycin. Cells were harvested, washed, and stained with annexin V and propidium iodide, according to the manufacturer's instructions (PharMingen). The cells were then analyzed on a FACSCalibur device. Dead cells were defined as annexin-V-positive cells.
Each subpopulation of thymocytes was stained with Cy-chrome-conjugated anti-CD4 MAb and phycoerythrin-conjugated anti-CD8 MAb (PharMingen) and washed. The cells were resuspended in 100 μl of PBS containing 4% paraformaldehyde, incubated for 20 min on ice, and washed. The cells were then resuspended in 50 μl of PBS containing 1% FBS, 0.1% sodium azide, 0.1% saponin (Sigma), and hamster anti-Bcl-2 MAb or isotype control Ab (5 μg/ml; PharMingen). After a 30-min incubation on ice, the cells were washed and further stained with fluorescein isothiocyanate-conjugated anti-hamster Ig (PharMingen) for 30 min on ice. The cells were then analyzed on a FACSCalibur device.
In vitro kinase assay.
To measure the p38MAPK activity, we used a p38MAPK kit (Cell Signaling Technology) according to the assay protocol of the kit. Briefly, DP thymocytes were stimulated for the indicated times and then lysed in lysis buffer. The supernatants of the lysates were immunoprecipitated with an immobilized p-p38MAPK MAb. The immunoprecipitated pellets were incubated in kinase buffer containing ATF-2 fusion protein and cold ATP. The reaction was terminated with SDS sample buffer. The samples were separated by SDS-polyacrylamide gel electrophoresis and analyzed for ATF-2 phosphorylation on Thr71 by Western blotting using a p-ATF-2 Ab and chemiluminescence detection.
Northern blot analysis.
Total RNA was extracted from thymocytes using TRIzol (Molecular Research Center, Inc.). For Northern blot analysis, 10 μg of total RNA was electrophoresed on a 1% agarose gel containing formaldehyde and then transferred to a Gene Screen membrane (NEN). Probe was labeled with [α-32
P]dCTP using a random primed DNA labeling kit (TAKARA SHUZO Co.). Prehybridization and hybridization were performed at 42°C in a solution containing 50% formamide, 5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2
, and 1 mM EDTA [pH 7.7]), 5× Denhardt's solution, 0.1% SDS, and salmon sperm DNA (20 ng/ml). Membrane was washed and subjected to autoradiography with a Bio-Image Analyzer BAS 1500 (Fuji Film). The probe DNAs for c-myc and glyceraldehyde-3-phosphate dehydrogenase cDNA were described previously (40