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note
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6 mitomycin C-treated syngeneic splenocytes and SLA (50 μg/ml) or 30 μM LACK synthetic peptide (amino acids 158 through 173).
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16
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10244242316
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note
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3 cells per well) in triplicate to 96-well microtiter plates (Millipore, Bedford, MA) that had been coated with the capturing monoclonal antibodies (mAbs) (2 μg/ml), antibody to IFN-γ, antibody to IL-4, or antibody to IL-5. After 24 hours, cells were removed and spot-forming cells were visualized with biotinylated detecting mAbs and avidin D peroxidase in conjunction with 3-amino-9-ethylcarbazole (Sigma) substrate. Spots were counted under a dissecting microscope, and the frequency of antigen-specific cells was determined from the difference between the number of spots seen with and without antigen.
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19
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1 mouse embryos and implantation into pseudopregnant foster mothers. The IE-LACK trangenic mice studied were backcrossed eight generations to BALB/c mice and were maintained under specific pathogen-free conditions.
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3H]thymidine during the last 18 hours of culture. Plates were harvested and counted on a Betaplate Reader (Pharmacia, LKB). Parallel cultures were set up to measure amounts of cytokines in the supernatants by enzyme-linked immunosorbent assay, with the use of specific mAbs to IL-4, IL-5, or IFN-γ (PharMingen, San Diego, CA).
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21
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10244228904
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note
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1 background, we cannot completely rule out the possibility that the transgene integrated near and segregated with a resistant gene despite eight generations of backcrossing to BALB/c mice. This is, however, very unlikely to happen.
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Single-letter abbreviations for the amino acid residues are as follows; A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr
-
Single-letter abbreviations for the amino acid residues are as follows; A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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10244264483
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note
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We thank K. Marcu, S. McSorley, and E. Sercarz for reviewing this manuscript; D. Mathis, C. Benoist, and C. Gabaglia for reagents; and F. Altare for preparing the IE-LACK transgene. Supported by grants from the Conseil Régional de la Région PACA, the Special Program for Research and Training in Tropical Diseases (United Nations Development Programme-World Bank-World Health Organization), and the Ministère de l'Education Nationale, de la Recherche et de l'Enseignement Supérieur (France).
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