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Blood, Vol. 114, Issue 7, 1355-1365, August 13, 2009
Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon
Blood Saint Fleur et al.
114: 1355
Supplemental materials for: Saint Fleur et al
Cyclon-Tg and Cyclon-deficient mice. The cDNA encoding HA-mCyclon1 was inserted in the VA-hCD2 cassette.2 After removing vector sequences, the resultant transgene was injected into pronuclei of fertilized eggs from FVB/N mice and backcrossed with C57BL/6 mice at least 6 times. The thymidine kinase gene followed by a short homology arm, enhanced green fluorescent protein (EGFP), a loxP-flanked neor cassette, and a long homology arm were cloned into the pTNLrSP4 vector to generate the targeting vector used for homologous recombination in embryonic stem cells. The short arm consisted of a 1.9-kb fragment located immediately 5′ of the ATG translational start site of Cyclon exon 1, and was amplified by PCR from tail DNA of a C57BL/6 mouse with a Nco I site at its 3′ end. The EGFP-coding sequence (BD Biosciences) was fused to the short arm through the Nco I site. The long arm consisted of 7-kb fragment located immediately 3′ of the ATG translational start site of Cyclon exon 1 and extending to Cyclon exon 4, and was amplified by PCR from tail DNA of a C57BL/6 mouse. Embryonic stem cells (iTL IC1; C57BL/6) were electroporated with the linearlized targeting vector, and 2 out of 300 G418-resistant colonies had the correctly targeted Cyclon locus. Electroporation of the targeting construct and blastocyst injection were done at ingenious Targeting Laboratory (iTL), Inc. Initial screening was done by PCR, amplifying 2.6-kb fragment starting 700 bp 5′ of the short arm in the Cyclon locus and ending at the 5′ end of EGFP. Correct insertion of the sequence encoding EGFP was also confirmed by Southern blot analysis (Fig. S7E and F). The neor-containing embryonic stem cells were injected into blastocysts and one chimeric male was obtained. Germline transmission of the mutant allele was obtained with this chimera and yielded Cyclon+∕EN mice. The genotype of the Cyclon+∕EN mice was established by PCR with primers surrounding the Cyclon exon 1 and EGFP: the 5′ primer, the 3′ primer, or the EGFP primer was 5′-CCCTTGCAAGAACTCGCAGTTTGCAAAAAACGCAT-3′, 5′-AAGGTTTTCTGGAGACAGAGGTTTTAGGCCTTCCA-3′, or 5′-ATGCCGTTCTTCTGCTTGTC-3′, respectively. REFERENCES 1. Hoshino A, Fujii H. Redundant promoter elements mediate IL-3–induced expression of a novel cytokine-inducible gene, cyclon. FEBS Lett. 2007;581:975–980.
2. Zhumabekov T, Corbella P, Tolaini M, Kioussis D. Improved version of a human CD2 minigene based vector for T-cell–specific expression in transgenic mice. J Immunol Methods. 1995;185:133–140.
Files in this Data Supplement:
- Figure S1. Transgenic expression of Cyclon does not restrict T-cell proliferation in vitro (JPG, 214 KB)
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Whole splenocytes were stained with 4 µM carboxy-fluorescin diacetate succinimidyl ester (CFSE) in 1× PBS, 0.1% BSA for 10 minutes at 37°C, activated with anti-CD3 Ab (10 µg/ml) for 4 days and stained with PE-conjugated anti-CD4 or anti-CD8 Abs for FACS analysis.
- Figure S2. Transgenic expression of Cyclon has no effect on FasL expression (JPG, 250 KB)
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CD4 (+) or CD8 (+) T cells sorted magnetically were activated with anti-CD3 Ab (2 µg/ml) and anti-CD28 Ab (2 µg/ml) for 2 days. Live cells were purified by density centrifugation and stained with PE-conjugated anti Fas Ab (dotted lines) or re-stimulated with 1 µg/ml anti-CD3 in the presence of IL-2 (5 ng/ml) for 24 hr and stained with anti-Fas Ab (solid lines).
- Figure S3. Fas expression on resting T cells from WT and Cyclon-Tg mice (JPG, 241 KB)
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CD4 (+) or CD8 (+) T cells were gated on splenocytes that were unstained or stained with anti–Fas-PE Ab.
- Figure S4. Transgenic expression of Cylon does not affect expression levels of FADD, FLIP, and caspase 8 (JPG, 319 KB)
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(A) CD4 (+) or CD8 (+) T cells from WT or Cyclon-Tg mice were activated with anti-CD3 Ab (2 µg/ml) and anti-CD28 Ab (2 µg/ml) for 2 days. Then, cells were re-stimulated with anti-CD3 Ab (1 µg/ml) in the presence of IL-2 (5 ng/ml) for 1 day. Whole-cell lysates were prepared from freshly isolated cells (activation 0), singly-activated cells (activation 1), or re-stimulated cells (activation 2) and subjected to immunoblot analysis with anti-FADD (sc-5559), anti-FLIPS∕L (sc-5276) or anti-caspase 8 Ab (sc-7890). Abs were purchased from Santa Cruz Biotechnology, Inc. (B) CD4 (+) T cells from IL-2Rα− or Cyclon-Tg IL-2Rα− mice were activated with anti-CD3 Ab (2 µg/ml) and anti-CD28 Ab (2 µg/ml) for 2 days in the presence of IL-15 (100 ng/ml) for 2 days. Whole-cell lysates were prepared from freshly isolated cells (activation 0) or activated cells (activation 1) and subjected to immunoblot analysis.
- Figure S5. Expression levels of Cyclon do not affect cell death induced by a non-specific cell death inducer (JPG, 118 KB)
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CD4 (+) or CD8 (+) T cells from WT or Cyclon-mutant mice were activated with anti-CD3 Ab (2 µg/ml) and anti-CD28 Ab (2 µg/ml) for 2 days. Live cells were purified by density centrifugation and cultured in puromycin (1 µg/ml or 5 µg/ml for CD4 (+) T cells or CD8 (+) T cells, respectively). Cells were incubated for 2 days, and viability was examined by PI staining and flowcytometry.
Additional supplemental figures can be found here.
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