Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice

Science

Volumn 273, Issue 5271, 1996, Pages 109-112

  Lau, Henry T ^a,d   Yu, Ming ^a   Fontana, Adriano ^b   Stoeckert Jr , Christian J ^c  

^a UNIVERSITY OF PENNSYLVANIA   (United States)

^b UNIVERSITY HOSPITAL ZURICH   (Switzerland)

^c CHILDREN S HOSPITAL OF PHILADELPHIA   (United States)

^d JOHNS HOPKINS HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALLOGRAFT; ANIMAL EXPERIMENT; ANIMAL TISSUE; ARTICLE; DIABETES MELLITUS; GRAFT REJECTION; MOUSE; MYOBLAST; NONHUMAN; PANCREAS ISLET; PANCREAS ISLET TRANSPLANTATION; PRIORITY JOURNAL;

ANIMALIA;

EID: 0030017476     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5271.109     Document Type: Article

References (25)

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11. Myoblasts were derived from thigh muscles of adult B6 mice 72 hours after treatment with 25 μl of 0.25% bupivacaine intramuscularly. Primary cultures were maintained as myoblasts in F10 medium supplemented with 20% (v/v) fetal bovine serum and basic fibroblast growth factor (2.5 ng/ml) (Promega, Madison, WI). Murine FasL or human Fas complementary DNA was cloned into the BCMGS Neo expression plasmid (6), and B6 myoblasts were transfected with 10 μg of plasmid DNA and 40 μg of lipofectamine (Gibco, Gaithersburg, MD) in 100-mm plates. Selection with G4 18 (200 μg/ml) (Gibco) was initiated 48 hours after transfection and continued for 2 weeks, after which resistant colonies were pooled and designated Bfasl or Bfas.
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23. 2 in phosphate-buffered saline (PBS) for 15 min. Nonspecific sites were blocked with 10% goat serum and 0.3% Triton X-100 in PBS for 1 hour, with addition of free avidin to block endogenous biotin followed by incubation for 30 min with free biotin. The sections were then incubated overnight at 4°C with undiluted culture supernatant from pFAS-Fcll-transfected Neuro-2a cells. Fas-Fc binding was detected by sequential application of biotin-conjugated goat antibodies to human immunoglobulin (Fc specific), streptavidin-conjugated horseradish peroxidase, and diaminobenzidine substrate solution (Zymed, San Francisco, CA). Sections were than counterstained with hematoxylin.
24. 2 in PBS for 15 min. Sections were then incubated with 10% goat serum in PBS for 1 hour, after which the 3′-OH ends of fragmented DNA were labeled by TUNEL with an in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany). Sections were then counterstained with eosin.
25. Care and use of all animals were in accordance with the guidelines of the animal care committee of the Children's Hospital of Philadelphia. We thank J. M. Templeton Jr. for his support; K. High and S. Surrey for critical review of the manuscript; and S. Nagata for reagents.