Figure 2.
Distribution of expression measurements after transient and site-directed stable transfection. (A) Partitioning DNA segments into classes of preCRMs or preNeutrals, based on RP score and predicted GATA-1 binding sites. (B) Transient transfection assay. (Left) Maps of the expression vectors show a firefly luciferase reporter gene expressed from the human A gamma globin gene promoter (HBG1pr) and a multiple cloning site (MCS) for inserting the preCRMs (pCRM). A co-transfection control plasmid has the Renilla luciferase gene expressed from the thymidine kinase promoter (TKpr). (Right) The distributions of luciferase activity measurements for three categories of tested DNA (preCRMcc, preCRMcnc and preNeutral) in transfected K562 cells. (C) Site-directed stable integration assay. The donor plasmid has an EGFP gene expressed from the human HBB promoter, preceded by a MCS into which preCRMs are inserted for test constructs. Cre-mediated exchange via loxP sites can replace the HyTK marker by the test expression cassette. The graphs show the distribution of EGFP fluorescence in pools of cells carrying expression constructs from the three major groups of tested DNAs, represented as log2 fold change, for each day during HMBA induction period. The distributions are depicted as box-plots, in which the box encompasses the values from the 25th to 75th percentiles, and the median is shown as a line across the box. The whiskers extend to the closer of two values: either the limit of the values in the distribution or 1.5 times the interquartile distance (the difference between the 75th and 25th percentiles). Values beyond 1.5 times the interquartile distance are plotted as circles.