Enhancer Function of Integrated Alb Reporters Figure 1. Cloning and recombination strategies. A: Rat Alb and its upstream region. Using restriction sites in the pLL1 linker, GFP was combined with Alb123, Region 1 (a NarI – XhoI segment from 213.7 to 23.9 kb), Region 2 (a segment from 23.9 to 20.2 kb, generated by PCR that added terminal FseI and PstI sites), and Alb123 (20.2 to the transcription start, with terminal PstI and BglII sites) in a series of reporter plasmids. The 16.5-kb Alb gene was cloned by joining a 4.4 kb proximal segment (transcription start to SmaI, amplified by PCR that added an SgfI site) and a 12.1 kb distal segment (SmaI – EagI). To insert the Alb123-GFP reporter as an FseI–SmaI segment, the Alb-containing plasmid was cut with FseI and SgfI, and blunted at the latter site. The resulting reporter plasmids ranged from 4–40 kb (Table S1). B: Schematic map of the 290 bp linker showing the restriction sites for locus assembly. C: Mechanism of RMCE. The targeting cassette encodes an HY-TK fusion protein that makes the cell sensitive to Gancyclovir. Integration of reporter constructs in pLL1 or pLL2 occurs via Cre-mediated recombination, and can take place in two orientations due to the inverted arrangement of the LoxP sites [32]. Loss of the HY-TK gene renders the integrant cells resistant to Gancyclovir. doi:10.1371/journal.pone.0023234.g001 enhancers. The 16.5-kb rat Alb gene has 15 exons and a single liver-specific enhancer (‘‘E1’’) at 210 kb, a remarkable distance at the time of its discovery [25]. Most E1 function has been mapped to a 200-bp minimal enhancer that binds FOXA and GATA4 [26,27,28,29,30]. Nevertheless, E1 only weakly stimulates tran- scription in transient assays [25,31]. This weakactivityraises intriguing questions, whether E1 needs integration in its natural context for proper function, and whether additional enhancers are required for optimum Alb expression. Our analysis characterizeda 35-kb region containing the full Alb gene and 18-kb upstream segment. The region includes E1 and several other conserved non- coding regions, potentially novel regulatory elements. We adapted an efficient system of recombinase mediated cassette exchange (RMCE) that uses inverted LoxP sites to prevent re-excision of the integrated transgene and allows insertion of DNA segments that do not contain a selection marker [32,33]. After incorporation of a targeting cassette into hepatocyte-like HuH7 cells, integration of Alb- gene reporters validated the experimental system by demonstrating that transcription of Alb is regulated by a much more complicated system of distant enhancers than revealed by previous studies. Results Transcriptionally permissive target loci We used human HuH7 cells to study Alb transcription controls because they express high levels of Alb mRNA like fetal and adult hepatocytes [34]. HuH7 cell clones with chromosomally integrat- ed targeting cassettes were isolated by hygromycin selection after transfection. Southern blot identified clones with single copy integration, which were then screened for efficiency of RMCE using a test plasmid. Two clones, HuH7-9 and HuH7-10, showed the mostefficient recombination and were used for integrating plasmids containing regions of the rat Alb combined with a GFP reporter (Figs. 1, S1; Table S1). To characterize their transcriptionally-permissive chromosome environments, we sequenced the integration sites of both clones. In PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23234