Cell lines
Human hepatoma cells HuH-7 (JCRB0403-A, JCRB Cell Bank, Japan) and HepG2 (ATCC® HB-8065), murine hepatoma cells Hepa 1–6 (C0015005, AddexBio, San Diego, CA), and HEK-293T (T0011002, AddexBio) cells were grown in DMEM (Gibco BRL) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 μg/mL streptomycin and 100 U/mL penicillin.
Construction of AAV plasmids
Synthetic DNA sequences of minimal BSEP promoters from mouse (imPr) and human (ihPr) origin (Additional file 1: Fig. S1) were based on human and mouse full-length BSEP promoters, respectively (NCBI Reference Sequences: AF190696.1 and AF190697.1, respectively) and obtained from GenScript (Nanjing, China) cloned into pUC57 flanked by Mlu I and Nhe I sites (pUC57-imPr & pUC57-ihPr). To generate AAV plasmids expressing luciferase, we used plasmid pAAV-A1AT-LucPEST (kindly provided by Dr. Tomas Aragon, Cima Universidad de Navarra, Spain), which contains AAV2 ITRs flanking the destabilized firefly luciferase sequence (LucPEST, GenBank accession number AY603759) downstream of the alpha-1 antitrypsin (A1AT) promoter. The DNA fragment containing each minimal promoter was extracted from pUC57-imPr and pUC57-ihPr by digestion with Mlu I and Nhe I and subcloned into pAAV-A1AT-LucPEST substituting the A1AT promoter by ihPr and imPr, generating in this way plasmids pAAV-ihPr-LucPEST and pAAV-imPr-LucPEST, respectively. The sequences of imPr in which one, three or five extra tandem repeats of the murine IR-1 element (TTAGGCCATTGACCTA), or three extra tandem repeats of the IR-1 element preceded by the ER2 motif (TGGACT) at the 5´end were ordered to Genscript and subcloned into pAAV-imPr-LucPEST plasmid replacing the imPr promoter using Kpn I and Sac I restriction sites, generating plasmids pAAV-imPr+1xIR-LucPEST, pAAV-imPr+3xIR-LucPEST, pAAV-imPr+5xIR-LucPEST, and pAAV-imPr+3xIR-ER2-LucPEST, respectively. An extended version of imPr including the liver receptor homolog-1 (LRH-1) element (TTTCTAAAGCT) at its 5´ end was also ordered to GenScript and subcloned into pAAV-imPr-LucPEST as described above, generating plasmid pAAV-imPr+LRH-1-LucPEST.
To generate pAAV-mBSEPpr-LucPEST, a 2488 nt synthetic sequence of full-length mouse BSEP promoter (NCBI AF190697.1) was also ordered from GenScript cloned into pUC57 flanked by Mlu I and Nhe I sites and used to substitute A1AT promoter in pAAV-A1AT-LucPEST, as described before (Additional file 1: Fig. S2a). An additional control vector containing the luciferase gene downstream of a full-length human BSEP promoter of 2563 nt (p-2563/+4-Luc) (Additional file 1: Fig. S2b) was kindly provided by Dr. James Boyer (Yale University, New Haven, CT). Finally, a plasmid expressing human FXRɑ1 (pcDNA3.1-hFXRα1) was ordered from GenScript and a plasmid expressing human FXRɑ2 (pCMV-hFXRɑ2) was obtained from cDNA Resource Center (Bloomsburg, PA).
Analysis of luciferase expression in vitro
Twelve-well plates were seeded with human (HepG2 or Huh-7) or mouse (Hepa 1-6) hepatic cells on day 0 with 5 × 105 cells/well and co-transfected the next day with 0.5 µg/well of the plasmid expressing luciferase to be tested and 0.1 µg/well of the plasmid expressing the selected FXR isoform using lipofectamine 2000 (Thermo Fisher, Waltham, Massachusetts) at a DNA/lipofectamine ratio of 1:3. To control the transfection efficiency, cells were co-transfected with 0.1 µg/well of a plasmid expressing Renilla-luciferase from a constitutive promoter (pRL-CMV, AF025843). At 24 h post-transfection, cells were incubated with 30 µM (human cells) and 100 µM (mouse cells) chenodeoxycholic acid (CDCA, Sigma-Aldrich, St. Louis, MO), or 0.3% and 1.0% DMSO (Sigma-Aldrich) as controls, for 30 h. The amount of CDCA used to induce each specific cell line was previously optimized (data not shown). The expression of the Renilla/firefly luciferase system was determined from cell lysates using a non-commercial dual luciferase enzyme assay as described Dyer et al. [28] and the results were measured with an Orion L Microplate Luminometer (Berthold Technologies, Germany).
Production of AAV vectors
To produce AAV8 viral particles (VPs), 150 cm2 flasks containing confluent HEK-293T cells were cotransfected, using 25 kDa linear polyethyleneimine (Polysciences, Warrington, PA), with the AAV plasmid of interest and pDP8.ape (Plasmid Factory, Germany), which contains adenoviral helper genes plus AAV2 rep and AAV8 cap genes. After 72 h, the supernatant was collected, treated with 8% v/v polyethylene glycol and incubated for 48–72 h at 4 °C. The supernatant was centrifuged at 1378g for 15 min and the pellet was resuspended in lysis buffer (50 mM Tris–Cl, 150 mM NaCl, 2 mM MgCl2, 0.1% Triton X-100) and stored at − 80 °C. Cells were also collected, treated with lysis buffer, and frozen at − 80 °C. After three cycles of freezing and thawing, the VPs obtained from the supernatants and cell lysates were purified by ultracentrifugation at 350,000g for 2.5 h in a 15–57% iodioxanol gradient. Finally, the purified virus was concentrated using Amicon Ultracel 100 K ultra centrifugal filters (Millipore). The AAV titers (VGs/mL) were determined by quantitative PCR (qPCR). VGs treated with DNase were extracted from VPs using the High Purity Viral Nucleic Acid Kit (Roche, Switzerland) and specific primers for LucPEST (Fw: 5′-TCTGAGGAGCCTTCAGGATT-3′ and Rv: 5′-TTTTGGCGAAGAAGGAGAAT-3′) and ITRs (Fw: 5′-GGAACCCCTAGTGATGGAGTT-3′ and Rv: 5′-CGGCCTCAGTGAGCGA-3′) were used for qPCR.
Animal studies
FVB Mdr2−/− mice (FVB.129P2-Abcb4tm1Bor; JAX stock #002539) (Abcb4−/−) [16] and FVB Mdr2+/+ (Abcb4+/+) mice (The Jackson Laboratory, Bar Harbor, ME) were raised in our own facilities (Cima Universidad de Navarra, Spain). C57BL/6 wild-type mice were purchased from Envigo (Spain). Mice were housed on a 12 h light–dark cycle and received water and food ad libitum using a standard food or a special diet according to the study. Treatment with AAV vectors was performed in male and female mice at four weeks of age by retroorbital injection. For serum analysis, mice were bled intravenously at weeks 1, 3, 5, 10, 14, 18, and 22 post-inoculation. Serum was separated from whole blood by centrifuging at 2300×g for 15 min in a microfuge. Total bile acids were quantified using a HITACHI C311 analyzer (Roche). In some experiments C57BL/6 mice were given a special diet containing 0.2% (w/w) sodium cholate (Sigma-Aldrich). This diet was administered in three-week periods alternating with three-week periods of normal diet, for a total of three cycles. All animal experiments and procedures were carried out in accordance with the ethical standards for animal experimentation and the studies were reviewed and approved by the Institutional Ethical Committee of the University of Navarra (protocol numbers: 082c-17 for reproduction, 086-17 and 024-18 for animal studies).
Vector genome and transgene expression quantification
Vector genome copies and LucPEST mRNA present in liver extracts were determined by qPCR using iQ™ SYBR® Green (BioRad, Hercules, CA) in a CFX96 Real-Time Detection System (BioRad) with primers specific for LucPEST (Fw: 5′-TCTGAGGAGCCTTCAGGATT-3′ and Rv: 5′-TTTTGGCGAAGAAGGAGAAT-3′). Mouse GAPDH was used as a normalizing gene (Fw: 5′-GGATGCAGGGATGATGTTC-3′ and Rv: 5′-TGCACCACCAACTGCTTA-3′).
Bioluminescence imaging of mice
For in vivo quantification of luciferase activity, mice were anesthetized via intraperitoneal injection with a mixture of ketamine, physiological saline and xylazine (in a ratio 3:2:3) and received an intraperitoneal injection of 100 μL d-luciferin potassium-salt substrate (Promega, Madison, Winconsin) dissolved in PBS at a final concentration of 30 µg/mL. Light emission was measured using a CCD luminometric camera (Biospace Lab, France). Photon counts were acquired 5 min after substrate administration. Light surface images were obtained immediately after each photon counting session to provide an anatomical view of the animals. Image processing and signal intensity quantifications were performed using M3 Vision software (Biospace Lab). Images are displayed as a pseudo-color photon count image, superimposed on a gray scale anatomic white-light image, allowing assessment of both bioluminescence intensity and its anatomical source. The number of photons emitted per second was calculated as a measure of luciferase activity utilizing a constant region of interest.
Statistical analysis
Data are presented as mean values ± standard error of the mean (SEM) and were statistically analysed using unpaired or paired t test with GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA) considering P < 0.05 significant.