Enhancer Function of Integrated Alb Reporters Figure 8. Sequence analysis of two new Alb enhancers. Left: E2, localized to a 784 bp region by deletion mapping. Right: the 974 bp minimal region containing E4. Both regions were aligned to the human sequence (DCODE, http://ecrbrowser.dcode.org/); bold, conserved bases; *, insertion in the rat gene; -, insertion in the human gene. Putative transcription factor binding sites were detected with the TRANSFAC database using the RVISTA utility of DCODE, and with binding site models compiled in our laboratory. Sites with high-rank matches by both analyses were annotated in the figure. The ENCODE tracks at the human Alb gene, based on the Human Mar. 2006 (NCBI36/hg18) Genome Assembly (http://genome.ucsc.edu/ cgi-bin/hgTracks), include studies of HepG2 cells which mapped chromatin hypersensitive regions and ChIP localizations of HNF4 and C/EBP binding. For both enhancers, the human regions collinear with the illustrated rat sequences have hypersensitive regions and bind HNF4 and C/EBP, although these localizations cannot be precisely mapped to individual binding sites in either genome. doi:10.1371/journal.pone.0023234.g008 Cell clones for targeted integration A 2.5 kb PvuII fragment containing the cassette pL1-HY-TK- 1L [32] was electroporated into HuH7 [34,46]cells and selected with 200 mg/ml of Hygromycin B. HygR colonies were main- tained under continuous antibiotic selection. Southern blot analysis (not illustrated) demonstrated fourclones that contained single cassette copies. These were screened by transfection of pL1- CMV-Neo-1L alone or in cotransfection with pCMV-Cre/ pBS185 [47], followed by selection with geneticin. The number of colonies was counted in each condition, and the difference between the two values represented the number of Cre-targeted integrations. Among the four clones, targeted integration ranged from 29–78 targeted integrations per 106 cells. HuH7-9 had the greatest efficiency of targeted integration and was used as the standard system for analysis of albumin gene constructs after an initial comparison with HuH7-10 (see Fig. 3 below). Cell culture and transfection HuH7-9 and HuH7-10 cells were maintained in Williams E medium supplemented with 1% L-Glutamine, 1% Pen/Strep, and 5% fetal bovine serum at 37uC in 5% CO2. For transient assays, 0.56106 cells were transfected with 2.5 mg of DNA in Lipofecta- mine (Invitrogen) according to the manufacturer’s protocol. Cells were fed with fresh medium at 24 hr, then trypsinized, washed, and analyzed by flow cytometry at 48 hr. For targeted integration, cells were reselected with 350 mg/ml hygromycin B solution for one or two passages before stable transfection. Then 16106 cells were transfected with 4 mg of gene constructs in pLL1 or pLL2 and 1 mg pCMV-Cre (Clontech), using Lipofectamine LTX with PLUS reagent (Invitrogen), on 60 mm plates. At 24 hr, cells were split 1:2 to 100 mm plates. 1 mM gancyclovir was added at 48 hr [33]. Discrete colonies with uniform fluorescence were isolated after 3–4 weeks of selection. Isolates were screened by PCR and Southern blots analysis [48]. To study the effects of growth arrest, control and GFP- expressing cell clones were seeded at low density (,25% confluence) and fed with Williams E medium containing 5% or 0.1% serum 24 hr after plating. Clones were analyzed by flow cytometry 48 and 96 hr following the change of medium. Genomic location of the targeting cassette Genomic DNA was digested with AvrII or PvuII and self-ligated at 1–2 ng/ml. Inverse PCR of the cassette-genomic junctions used primers Target F and Target R. PCR products (1–4 kb) were sequenced using the same primers. Flow Cytometry 1–26106cells were trypsinized, washed, resuspended in cold PBS containing 1% FBS, and analyzed with a FACSCalibur flow cytometer (Becton-Dickenson, Franklin Lakes, NJ). Forward and side scatter measurements were used to differentiate live and dead cells. Measurements for 20,000 cell events in stable clones, or 60,000 cell events in transient transfection assays, were further analyzed with FCS Express software (De Novo Software, Los Angeles). For transient assays, average fluorescence intensity was determined by histogram subtraction of the distribution deter- mined for untransfected cells from distribution of transfected cells. Since all transfections utilized the same DNA concentration of PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23234