Enhancer Function of Integrated Alb Reporters Figure 7. Effects of growth on enhancer function. A: Selected flow cytometry comparisons. Cells were plated at 25% confluence in 5% fetal calf serum, and allowed to reach confluence (,72 hr) or switched to 0.1% serum after 24 hr. Exponential growth at 48 hr in serum-containing medium (dotted lines) was compared to growth arrest (solid lines) due to cell confluence at 96 hr (left), or serum starvation at 48 and 96 hr (center and right). A control curve for untransfected cells appears at the left of each panel (solid lines). B: Stimulation of O1 clones by growth arrest. A series of clones systematically resolved the effects onintact Regions 1, 2, ALB, and Enhancers E1 and E2. C: Stimulation ofO2 clones by growth arrest. A more limited series of O2 clones was evaluated as above. B, C; the plots show mean and standard deviation of fluorescence intensity averaged from two separate experiments. Expression was normalized to the level observed for growth at 48 hr in serum. doi:10.1371/journal.pone.0023234.g007 with these observations, because proliferating hepatocytes or cell lines have lowerexpression than the quiescent hepatocytes of normal liver [34,35]. Indeed, growth arrest strongly stimulated gene expression, particularly of constructs containing E2. The next stepsare to work out the mechanism of this specific effect, along with a general characterization of the dissolution and reassembly of looping during the cell cycle. Our system of targeted integration and Alb gene constructs will provide an ideal platform for this research. Materials and Methods Plasmid constructs Cloning vectors Locus Linker 1 and 2 (pLL1, pLL2) were constructed for assembly of restriction enzyme fragments excised from genomic clones of the rat Alb-Afp locus, and for Cre-mediated recombination into mammalian cells (Fig. 1, Table S1). A 290 bp synthetic linker (Figs. 1B and S1) was inserted between the NdeI and HindIII sites of pUC19 (pLL1) and also transferred to pBR322 (pLL2). The low copy number of the latter facilitates cloning of large genomic DNA segments. The plasmids incorporate the following features. Two loxP sites in opposite orientation allow Cre-mediated insertional recombination with the targeting cassette. Two I-SceI sites flank the cloning linker to allow incision of integrated gene segments. Unique restriction sites that do not cut the Alb-Afp locus (BssHII, FseI, SgfI, BsiWI, SgrAI, and SfiI), or cut the entire locus only once (SmaI, EagI, and SacII), were arranged so that individual regions could be joined or excised through simple forced directional cloning strategies. Other restriction enzyme sites were arranged to facilitate locus assembly or to provide general cloning sites. GFP was amplified by PCR from pEGFP-N1 (Clontech, Mountain View, CA) with primers that added terminal SgfI and SmaI sites (Table S2) and cloned into pLL1 at those sites. The synthetic Alb123 promoter [45] was inserted between linker PstI and BglII sites. For cloning of large DNA segments, restriction fragments were resolved on 0.5% agarose gels stained with SYBR gold (Invitrogen, Carlsbad, CA), detected with a Dark Reader Transilluminator (Clare Chemical Research, Denver, CO), and purified using a QIAEX II gel extraction kit (Qiagen, Hilden, GER). Ligations, performed with standard procedures, were electroporated into DH10B (Invitrogen) or SURE (Stratagene, La Jolla, CA). Deletion mapping of enhancer-containing regions was carried out with BstXI, ApaI, SgfI, or SmaI, or withspecific PCR primers (Table S2). PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23234