A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors
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G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.
KeywordsMouse artificial chromosome SIM system Split luciferase GPCR PTHR2 hEP4 ARRB1
Mammalian artificial chromosome vectors, including the human artificial chromosome (HAC) (Kazuki et al. 2011) and mouse artificial chromosome (MAC) (Takiguchi et al. 2012), are suitable systems for stable expression of multiple genes (Oshimura et al. 2014). We previously reported the proof-of-concept of a new system for sequential integration of multiple vectors (SIM) via Cre-loxP recombination and phage integrase/attachment sites, i.e., Bxb1 integrase/attB/attP and PhiC31 integrase/attB/attP, on a HAC (Suzuki et al. 2014). The SIM system is promising for investigation of multiple gene interactions based on circular plasmid vectors.
Materials and methods
First, the NELuc-ARRB1 expression vector, loxP_BxbiP_3HPRT_inspB4_PtNG415-ARRB1ins, was constructed by the following steps: The Vec0 fragment was prepared by digestion with SpeI and BsrGI. The fragment was ligated to inspB4ins that was digested with Acc65I and NheI. This vector was named loxP_BxbiP_3HPRT_inspB4ins. Then, loxP_BxbiP_3HPRT_inspB4ins was digested with MluI and SnaBI, and ligated to the fragment of pcDNA3.1(+)_myc-HisB_PtGRN415-ARRB1 that was prepared by digestion with MluI and PvuI. This vector was named loxP_BxbiP_3HPRT_inspB4_PtNG415-ARRB1ins. Second, the PTHR2-CEluc expression vector, pBG2-v1b1 ins PTHR2 ins, was constructed by the following steps. The pBG2-v1b1 fragment (Suzuki et al. 2014) was prepared by digestion with SpeI and MluI. The fragment was inserted into inspB4ins that was digested with NheI and AscI. This plasmid was named pBG2-v1b1_inspB4ins. Then, the two fragments of PTHR2-linker20-PtGRC394 in pcDNA4 V5_His(B) digested with BglII and EcoRI or EcoRI and PvuII were inserted into pBG2-v1b1_ inspB4ins that was digested with BamHI and SnaBI. This plasmid was named pBG2-v1b1_ ins PTHR2 ins. Third, the hEP4-CEluc expression vector, pBG2-v1b1 ins hEP4 ins, was constructed by the following steps: pBG2-v1b1_inspB4ins was digested with BamHI and SnaBI and ligated to hEP4-linker20-PtGRC394 in the pcDNA4 V5_His(B) fragment digested with BglII and PvuII (pBG2-v1b1_ ins hEP4 ins). The Bxb1-expression vector was constructed as previously described (Yamaguchi et al. 2006).
CHO cells containing MAC4 (CHO/MAC4) were cultured in Ham’s F12 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Biowest, Vieux Bourg, France), 1% penicillin/streptomycin (Wako), and 800 µg/mL hygromycin B (Wako) (Takiguchi et al. 2012; Narai et al. 2015). CHO/MAC4 cells containing a reconstructed hypoxanthine–guanine phosphoribosyl transferase (HPRT) gene and desired genes were selected in Ham’s F12 medium with 2% hypoxanthine-aminopterin-thymidine (HAT) medium (Sigma-Aldrich, St. Louis, MO, USA). HEK293 cells purchased from the American Type Culture Collection (catalog number CRL-157, Manassas, VA, USA) were cultured in Eagle’s minimum essential medium (MEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% MEM non-essential amino acids (Thermo Fisher, Waltham, MA, USA), and 1% l-glutamine (Thermo Fisher). HEK293 cells containing MAC4 with the desired genes were selected in Eagle’s MEM containing 200 µg/mL hygromycin B.
Construction of a hemagglutinin-conjugated anti-CD9 single chain antibody (ScFv) derived from the measles virus
As HEK293 cells were analyzed by flow cytometric analysis for detecting the expression of CD9 and CD46, microcell-mediated chromosome transfer (MMCT) with measles virus envelop protein (MV-MMCT) was applied and retargeted the trophism by fusion of an ScFv against CD9 (Katoh et al. 2010; Hiratsuka et al. 2015).
MMCT via hemagglutinin and a fusion protein derived from the measles virus
A total of 1 × 107 donor CHO cells were co-transfected with 12 µg of pTNH6-H-αCD9 and 12 µg of pCAG-T7-F using Lipofectamine 2000 (Thermo Fisher), according to the manufacturer’s instructions. Twenty-four hours after the transfection, the transfected cells were expanded in three T-25 flasks (Thermo Fisher) with Ham’s F12 medium for cell culture. After another twenty-four hours from the expansion, the transfected cells were cultured in Ham's F12 medium containing 20% FBS and 0.1 µg/mL colcemid (Thermo Fisher) at 37 °C for 48 h to induce micronucleation. The culture medium was changed, and the cells were incubated for another 24 h. The T-25 flasks containing CHO cells with micronuclei were filled with Dulbecco’s modified Eagle’s medium containing 10 µg/mL cytochalasin B (Sigma-Aldrich) and then centrifuged for 1 h using an Avanti HP-26XP, JLA-10,500 rotor (Beckman Coulter Life Sciences, Indianapolis, IN, USA) at 11,900×g to form microcells. The pellet including microcells was collected and filtered through 8-, 5-, and 3-µm pore size filters to purify the microcells. Microcell pellets were collected by centrifugation at 760×g in a table-top centrifuge (Kubota Corporation, Tokyo, Japan). To introduce the MAC into HEK293 cells, 2 × 106 HEK293 cells were cultured in a 6-cm dish (Corning, Corning, NY, USA). The purified microcells were co-cultured with HEK293 cells. After 24 h of the co-culture, the HEK293 cells were subcultured into three 10-cm dishes. The next day, drug selection was started with 200 µg/mL hygromycin B. About 21 days later, drug-resistant colonies were picked up and expanded for the following analyses.
Fluorescence in situ hybridization (FISH)
Metaphase chromosomes were prepared from colcemid-treated cell cultures by hypotonic treatment with 0.075 M KCl and methanol/acetate (3:1) (Wako) fixation. FISH was carried out using mouse Cot-1 DNA labeled with digoxigenin (Roche, Basel, Schweiz) and the inserted plasmid vector, loxP_BxbiP_3HPRT_inspB4_PtNG415-ARRB1ins, and pBG2-v1b1_ ins PTHR2, which were targeted to the chromosome fragment labeled with biotin (Roche). The DNA probes were labeled with a nick translation kit (Roche). Digoxigenin-labeled DNA probes were detected with an anti-digoxigenin-rhodamine complex (Roche), and the biotin-labeled DNA was detected using avidin-conjugated fluorescein isothiocyanate (Roche). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich). Metaphase images were captured digitally with a CoolCubeI CCD camera mounted on a fluorescence microscope (Axio Imager, Z2; Carl Zeiss, Oberkochen, Germany). Images were processed using ISIS software provided with the microscope.
DNA transfection for insertion of plasmid vectors using the SIM system
The ARRB1 expression vector, loxP_BxbiP_3HPRT_inspB4_PtNG415-ARRB1ins, was inserted into MAC4. Then, 2 × 106 CHO/MAC4 cells were transfected with 8 µg loxP_BxbiP_3HPRT_inspB4_PtNG415-ARRB1ins and 1 µg Cre-expression vector (Invitrogen, Carlsbad, CA, USA) in a 6-cm dish using Lipofectamine 2000. After 24 h, the transfected cells were subcultured into six 10-cm dishes and incubated for a further 24 h. Then, 2% HAT medium was added to select cells with reconstitution of the HPRT gene. About 14 days later, drug-resistant clones were picked up and expanded for the following analyses.
pBG2-v1b1_ins PTHR2 was transfected into cells containing ARRB1-MAC4. Then, 2 × 106 cells in a 6-cm dish were transfected with 8 µg pBG2-v1b1_ ins PTHR2 and 1 µg Bxb1 integrase expression vector using Lipofectamine 2000. After 24 h, the transfected cells were selected in medium containing 800 µg G418 (Promega, Madison, WI, USA) and 2% hypoxanthine-thymidine medium (Sigma-Aldrich) to decrease cytotoxicity of aminopterin remaining in the cells. About 15 days later, drug-resistant clones were picked up and expanded for the following analyses.
Luciferase complementation assay (LCA)
A total of 6 × 104 cells were expanded in each well of 96-well plate. The cells were stimulated with a GPCR ligand expressed in HEK293 cells. Then, the medium was removed, and the cells were cryopreserved at − 80 °C. Measurement of luciferase activity was performed with Emerald Luc Luciferase Assay Reagent Neo (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. Bioluminescence was detected by an EnVision (PerkinElmer, Waltham, MA, USA). Time-lapse analysis measured the bioluminescence every 5 min. Each well was measured three times and data were corrected for average bioluminescence activity, and the data were expressed as means ± Standard Error (SE). Student’s t-test was used to determine statistically significant differences.
Genomic PCR analysis
DNA was extracted with a Gentra Puregene cell kit (Qiagen, Germantown, MD, USA). PCR analysis was performed with ExTaq or LA Taq kits (TAKARA Bio Inc, Kusatsu, Japan). The following primer pairs were used for detection of each amplicon. HPRT junction: Trans-L1 (5′-TGGAGGCCATAAACAAGAAGAC-3′) and Trans-R1 (5′-CCCCTTGACCCAGAAATTCCA-3′); ARRB1: GPCR ARRB1 Fw (5′-ACGCACAGAATTCCGCTTGTGGATCTT-3′) and GPCR ARRB1 Rv (5′-GGCAACGAGTCCCTTTCCTACCAGGAG-3′); Bxb1 SIM junction: SIM HPRT Fw (5′-TGGAGGCCATAAACAAGAAG-3′) and SIM Neo Rv (5′-CGCCTTGAGCCTGGCGAACA-3′); PTHR2-CEluc: PTHR2 Fw (5′-GGAGCAGATTGTCCTTGTGCTGAAAGC-3′) and PTHR2 Rv (5′-CACGTTCCTGGGGATAGAGTCCACGAA-3′); hEP4-CEluc: hEP4 Fw (5′-CTCTGGGTTCCAGGTTCCACTGGTGAC-3′) and hEP4 Rv (5′-ATCTTGCCTGTCACGTTCCTGGGGATA-3′).
Construction of ARRB1-MAC4 in CHO cells and evaluation of the LCA in HEK293 cells
Construction of ARRB1-PTHR2-MAC4 in CHO cells and evaluation of the LCA in HEK293 cells
In addition to construction of ARRB1-PTHR2-MAC4, hEP4-CEluc and Bxb1 integrase expression vectors were transfected into CHO cells containing ARRB1-MAC4 (Fig. 1b, c). Two clones were picked up and confirmed to have correct plasmid insertion by PCR analysis. FISH analysis showed that the two clones maintained a single ARRB1-hEP4-MAC4 independently from host chromosomes (Fig. 3f). Next, ARRB1-hEP4-MAC4 was transferred into HEK293 cells by MV-MMCT, and 12 clones were obtained after drug selection with hygromycin B. Nine clones among the 12 clones were found to maintain the correct ARRB1-hEP4-MAC4 by PCR analysis. Eight clones proliferated well and were subjected to FISH analysis. These clones maintained a single ARRB1-hEP4-MAC4 independently from host chromosomes. The LCA was performed in the eight clones with 1 µM PGE2 for 20 min. The results showed that six clones emitted bioluminescence in response to the PGE2 stimulation. Among the analyzed clones, #2-6 showed the highest photon counts of ~ 35,000 and an S/B ratio of ~ 7.0. Time-lapse measurement of bioluminescence confirmed the reaction against PGE2 stimulation throughout the time course (Fig. 3h).
In conclusion, LCAs for two GPCRs were developed using a split luciferase and SIM system for gene loading into a MAC.
Previously, we reported a SIM system in which three fluorescent protein genes were inserted into a HAC. Here, we report the use of the SIM system with a MAC to develop LCAs for GPCR-ARRB1 insertion and chromosome transfer of the MACs into HEK293 cells for practical application of LCAs. Because ARRB1 is generally known as a counterpart of various GPCRs, an LCA for other GPCRs can be developed by simple insertion of a targeted GPCR-CEluc into ARRB1-MAC4 using the SIM system. Therefore, we inserted an ARRB1 expression unit in the first step (Fig. 2). ARRB1-MAC4 was transferred into HEK293 cells, and the function of ARRB1 was confirmed by transfection of a plasmid expressing SSTR2, which had already been used for the LCA (Fig. 2c) (Misawa et al. 2010). Each GPCR-CEluc expression unit was inserted based on ARRB1-MAC4 in CHO cells and transferred into HEK293 cells (Fig. 3). Thus, the LCAs for PTHR2 and hEP4 were performed in HEK293 cells (Fig. 3e, h).
The constructed MAC can be transferred to various cell lines. This is the first report of chromosome transfer from CHO cells to HEK293 cells. To introduce the MAC into HEK293 cells, 2 × 106 HEK293 cells were cultured in a 6-cm dish. Then, we obtained eight clones of HEK293 ARRB1-MAC4, 10 clones of HEK293 ARRB-1-PTHR2-MAC4, and nine clones of HEK293 ARRB1-hEP4-MAC4. Thus, the MMCT efficiency was ~ 2 × 10−5 for each chromosome transfer experiment.
Generally, random integration of an expression vector often leads to unpredictable amplification of the copy number and disruption of the expression cassette, resulting in various expression levels of unexpected patterns and even silencing. In this system, long-term gene expression was maintained with higher expression levels as a function of passages (Fig. 3e). Because the selectable marker and GPCR expression unit were loaded closely, these genes may relate to each gene expression level via the chromatin structure. Therefore, it is possible that cells with higher gene expression levels of selectable markers and the GPCR unit were enriched during long-term culture.
Each result of the LCA for ARRB1, PTHR2, or hEP4 showed different sensitivities. The HEK293 cell clones, which were transferred with each MAC (ARRB1, PTHR2, or hEP4), showed different sensitivities in the LCA (Figs. 2c, 3c, h). Because the host HEK293 cells were re-cloned via MMCT, the mRNA expression and protein synthesis ability of the host cells may be different from each clone. Therefore, clones showing higher expression levels in the LCA should be screened for practical use.
A MAC and MMCT enable introduction of an identical gene delivery vector independently from host chromosomes without the influence of chromatin modification from integrated sites on host chromosomes (Oshimura et al. 2014). Therefore, this technique allows a more accurate comparison of GPCR activities between cell lines.
This system includes a further gene integration site, ϕC31 attP, which can be recombined with ϕC31 attB by ϕC31 integrase. Importantly, the Bxb1 attB/attP and ϕC31 attB/attP reaction is irreversible unlike the Cre-loxP system. The gene-loading system using the SIM system with a HAC/MAC can be used in not only CHO cells but also HEK293 cells, although knockout of the HPRT gene in HEK293 cells is required. This system is useful to introduce multiple genes into a desired cell line by generation of the cell line containing the MAC in advance. Thus, the SIM system is convenient for gene loading of multiple genes into MAC and development of a general GPCR-ARRB1 complementation assay using a split luciferase with simultaneous loading in a single MAC.
We thank Nakamura for providing pTNH6-H and p-CAG-T7-F plasmids, and Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was supported in part by the Regional Innovation Strategy Support Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (M.O.).
N.U. conceived and designed the experiments. S.K. and T.S. constructed plasmid vectors for the SIM system to load ARRB1 and GPCRs. G.K. generated the ScFv against CD9 and constructed the plasmid vectors. S.K. constructed MACs, and performed PCR and FISH analyses, T. F. performed LCAs. N.U., M.S., Y.K., and M.O. wrote the manuscript.
The Chromosome Engineering Research Center (CERC) has a collaboration with Carna Biosciences, Inc. related to this project. CERC received funding from Carna Biosciences, Inc.
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