The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs).
Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.
Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America.
Ning
N
Wang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China; Department of Laboratory Medicine, the Affiliated Hospital, Binzhou Medical University, Yantai, Shandong, China.
Wenwen
W
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Jing
J
Cui
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Hongmei
H
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America.
Xiang
X
Chen
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Ruidong
R
Li
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Ningning
N
Wu
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Xian
X
Chen
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Sheng
S
Wen
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Junhui
J
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Liangjun
L
Yin
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Department of Cell Biology, Third Military Medical University, Chongqing, China.
Fang
F
Deng
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Department of Orthopaedic Surgery, Xiang-Ya Hospital of Central South University, Changsha, China.
Zhan
Z
Liao
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Department of Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China.
Zhonglin
Z
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Qian
Q
Zhang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Zhengjian
Z
Yan
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Wei
W
Liu
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; School of Bioengineering, Chongqing University, Chongqing, China.
Jixing
J
Ye
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Youlin
Y
Deng
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Zhongliang
Z
Wang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Min
M
Qiao
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America.
Hue H
HH
Luu
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America.
Rex C
RC
Haydon
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America.
Lewis L
LL
Shi
Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China.
Houjie
H
Liang
Molecular Oncology Laboratory, Department of Orthopaedic Surgery, The University of Chicago Medical Center, Chicago, Illinois, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
Tong-Chuan
TC
He
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis* / Q000235:genetics; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D004251:DNA Transposable Elements; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D013539:Simian virus 40
D000952:Antigens, Polyomavirus Transforming; D004251:DNA Transposable Elements