TY - JOUR
T1 - Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines
AU - Donai, Kenichiro
AU - Kuroda, Kengo
AU - Guo, Yijie
AU - So, Kyoung Ha
AU - Sone, Hideko
AU - Kobayashi, Masayuki
AU - Nishimori, Katsuhiko
AU - Fukuda, Tomokazu
N1 - Funding Information:
We thank Gustavo Mostoslavsky (Boston University School of Medicine) for providing the STEMCCA–loxP lentiviral vector. We also thank Akane Inagaki and Shizu Hidema (Graduate School of Agricultural Science, Tohoku University) for their helpful discussions. This work was supported by research grants from the Urakami Foundation , Daiwa Securities Health Foundation , and Asahi Group Foundation as well as a Japan Society for the Promotion of Science (JSPS) grant (KAKENHI, 23650587).
Funding Information:
293T cells and mouse embryonic fibroblasts (MEFs) were cultured in MEF medium: Dulbecco’s modified Eagle’s medium (DMEM, cat. no. 08459-35, Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (cat. no. 12483-020, Invitrogen, Carlsbad, CA, USA) and 100× Antibiotic–Antimycotic Mixed Solution (cat. no. 02892-54, Nacalai Tesque). Infected MEFs were selected for 9 days with DMEM containing 5% fetal bovine serum and 1.2 mg ml − 1 G418. Mouse iPS cells were cultured on MEF feeder cells in mouse iPSC medium: DMEM with 15% Knockout Serum Replacement (cat. no. 10828-028, Invitrogen), 0.22 mM 2-mercaptoethanol (cat. no. 21438-82, Nacalai Tesque), 100× MEM Nonessential Amino Acids Solution (cat. no. 139-15651, Wako Pure Chemical Industries, Osaka, Japan), 100× Antibiotic–Antimycotic Mixed Solution, and 1000× Leukemia Inhibitory Factor (human, recombinant, culture supernatant, cat. no. 125-05603, Wako Pure Chemical Industries). From the end of the infection to selecting colonies, low-molecular-weight compounds—1.5 μM CHIR99021 (cat. no. 163-24001, Wako Pure Chemical Industries), 0.5 μM PD0325901 (cat. no. 13034, Cayman Chemical, Ann Arbor, MI, USA), and 0.5 μM thiazovivin (cat. no. 04-0017, Stemgent, Cambridge, MA, USA)—were also added to the mouse iPSC medium as reprogramming enhancers [20] . The human iPS cell line was maintained on MEF feeder cells in human iPSC medium: DMEM/F12 GlutaMAX (cat. no. 10565-018, Invitrogen) with 15% Knockout Serum Replacement, 0.22 mM 2-mercaptoethanol, 100× MEM Nonessential Amino Acids Solution, 100× Antibiotic–Antimycotic Mixed Solution, and 4 ng ml − 1 basic fibroblast growth factor. A human iPS cell line (201B7) was provided by the RIKEN BioResource Center through the Project for Realization of Regenerative Medicine and the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT, Japan) [21] . MEFs were isolated from the embryonic tissues of C57BL/6JJcl mice (CLEA Japan, Tokyo, Japan) at embryo day 13.5. For feeder cell preparation, confluent MEFs were treated with mitomycin C (cat. no. M0503-2G, Sigma–Aldrich, St. Louis, MO, USA) at a concentration of 10 μg ml − 1 for 2.25 h and washed twice in phosphate-buffered saline (PBS). Feeder cells were seeded at a density of 2 × 10 6 cells per plate. The cells used in these experiments were all incubated at 37 °C under 5% CO 2 . All cells were trypsinized using 0.05% trypsin (1:10 dilution, cat. no. 35556-44, Nacalai Tesque). Animal experiments and related activities were approved by the Center for Laboratory Animal Research at Tohoku University.
PY - 2013
Y1 - 2013
N2 - Induced pluripotent stem (iPS) cells have proven to be an effective technology in regenerative medicine; however, the low efficiency of reprogramming is a major obstacle to the successful generation of iPS cell lines. One of the most important characteristics of a high-quality iPS cell line is the inactivation of transgenes driven by a retrovirus-derived long terminal repeat promoter. In this study, we established a novel marker system containing three kinds of proteins: secreted-type luciferase (MetLuc), copepod Pontellina plumata green fluorescent protein (copGFP), and an antibiotic-resistant gene product (Neor). The introduction of MetLuc-copGFP-Neor in mouse embryonic fibroblasts (MEFs) allowed us to monitor the reporter expression changes as an indicator of the state of silencing during reprogramming. Transformation of iPS cells induced a remarkable reduction in reporter activity, indicating that the retroviral silencing was detected successfully. Our system enables us to monitor the silencing status of transgenes and to efficiently select iPS cell lines that can be used for further applications.
AB - Induced pluripotent stem (iPS) cells have proven to be an effective technology in regenerative medicine; however, the low efficiency of reprogramming is a major obstacle to the successful generation of iPS cell lines. One of the most important characteristics of a high-quality iPS cell line is the inactivation of transgenes driven by a retrovirus-derived long terminal repeat promoter. In this study, we established a novel marker system containing three kinds of proteins: secreted-type luciferase (MetLuc), copepod Pontellina plumata green fluorescent protein (copGFP), and an antibiotic-resistant gene product (Neor). The introduction of MetLuc-copGFP-Neor in mouse embryonic fibroblasts (MEFs) allowed us to monitor the reporter expression changes as an indicator of the state of silencing during reprogramming. Transformation of iPS cells induced a remarkable reduction in reporter activity, indicating that the retroviral silencing was detected successfully. Our system enables us to monitor the silencing status of transgenes and to efficiently select iPS cell lines that can be used for further applications.
KW - Gene silencing
KW - Induced pluripotent stem cells
KW - Reporter system
KW - Reprogramming
UR - http://www.scopus.com/inward/record.url?scp=84884918495&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84884918495&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2013.08.014
DO - 10.1016/j.ab.2013.08.014
M3 - Article
C2 - 23973628
AN - SCOPUS:84884918495
VL - 443
SP - 104
EP - 112
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -