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The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts
Ning Wang1, Wenwen Zhang2, Jing Cui3
1Department 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.
Abstract:
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.
Insights
Mouse embryonic fibroblasts (MEFs) were effectively immortalized using piggyBac transposon technology. The resulting immortalized cells (piMEFs) maintain long-term proliferation and multipotency, crucial for research applications.
Area of Science:
- Cell Biology
- Stem Cell Biology
- Molecular Biology
Background:
- Mouse embryonic fibroblasts (MEFs) are multipotent progenitor cells with limited in vitro lifespan.
- Limited MEF lifespan hinders basic research and translational applications.
- Immortalization strategies are needed to overcome MEF proliferative limitations.
Purpose of the Study:
- To investigate the efficacy of piggyBac transposon-mediated SV40 T antigen expression for MEF immortalization.
- To assess if immortalized MEFs (piMEFs) retain multipotency and proliferative capacity.
- To evaluate the reversibility of immortalization and its impact on cell function.
Main Methods:
- Utilized the piggyBac vector MPH86 for SV40 T antigen expression in MEFs.
- Assessed proliferative activity and long-term cell culture of immortalized MEFs (piMEFs).
- Evaluated multipotency through in vitro differentiation assays (osteogenic, chondrogenic, adipogenic) and in vivo stem cell implantation studies.
- Reversed immortalization using FLP recombinase to remove SV40 T antigen.
Main Results:
- Efficient immortalization of MEFs was achieved using piggyBac transposon-mediated SV40 T antigen expression.
- piMEFs demonstrated enhanced proliferative activity and sustained long-term proliferation.
- piMEFs retained multipotency, differentiating into bone, cartilage, and adipose lineages in vitro and in vivo.
- Reversal of immortalization by FLP recombinase diminished differentiation potential, linked to reduced progenitor expansion.
Conclusions:
- PiggyBac transposon-mediated SV40 T antigen expression is an effective strategy for immortalizing MEFs.
- Reversibly immortalized piMEFs maintain long-term proliferation and multipotency.
- PiggyBac transposition offers a safe and efficient method for immortalizing progenitor cells from limited sources for research and clinical applications.
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