Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease
Figure Lengend Snippet: Transgenic mRNA and protein expression. ( A ) Design of primers to detect spliced hybrid GAD-HGH RNA (see text). ( B ) Reverse transcription–PCR analysis of tissues from tg(+) mice ( + ) and non-tg(+) mice (−) mice, in the presence (RT+) or absence (RT−) of avian myeloblastosis virus reverse transcriptase (AMV-RT) by using transgene-specific primers ( Upper ) and hypoxanthine-guanine phosphoribosyltransferase-specific primers ( Lower ). M, 100-bp molecular marker (GIBCO/BRL); B, brain; K, kidney; L, liver; T, thymus; H, heart; I, islets. The origin of the different bands is discussed in the text. Tissue lysates from adult ( C and D ) or day 2 ( E ) tg(+) mice ( + ) and non-tg(+) mice (−) were immunoprecipitated with Stiff Man Syndrome patient serum and Western blotted with mAb mN65 against GAD65 ( C and E ). The membrane in C was stripped and reprobed with GAD65-specific rabbit polyclonal antisera, 7673 ( D ). Sizes of proteins were estimated from coelectrophoresed, prestained markers (not shown).
Article Snippet: Five micrograms of tissue RNA extracted with guanidium isothiocyanate/phenol (RNAzol, Cinna/Biotecx, Houston, TX) were primed with random hexamers, reverse-transcribed with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim), and equal amounts (quantified by spectrophotometry) amplified by PCR.
Techniques: Transgenic Assay, Expressing, Polymerase Chain Reaction, Mouse Assay, Marker, Immunoprecipitation, Western Blot