Transcriptional gene silencing in a subset of lines. (A) Real time polymerase chain reaction (PCR) analyses of human and mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression in transgenic T cells. GM-CSF expression in spleen-derived T cells activated for 2 days in the presence of ConA, and then cultured for 2 days in the absence of ConA and re-stimulated for 4 h with 20 ng/ml phorbol myristate acetate (PMA) and 2 μM A23187. Values are expressed relative to mouse glyceraldehyde phosphate dehydrogenase (GAPDH) and divided by transgene copy number. Error bars indicate standard deviation. (B) chromatin immunopreciptation (ChIP) assay of the elongating Ser 2 phosphate form of RNA polymerase II performed on T lymphoblasts prepared from mouse lines A127 (open boxes) and J253 (closed boxes) before and after stimulation for 4 h with 20 ng/ml PMA and 1 μM A23187. This panel depicts a representative experiment but we have obtained similar results in an independent analysis of lines M268 and D184. (C) Time course of human GM-CSF induction in spleen cells activated with 20 ng/ml PMA and 2 μM A23187.