The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs)

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.

Functional Characteristics of Reversibly Immortalized Hepatic Progenitor Cells Derived from Mouse Embryonic Liver

“…Using the reversible immortalization system expressing SV40 T antigen [49], we have immortalized several types of progenitors and adult cells, including MEFs, mouse cardiomyogenic progenitor cells, mouse articular chondrocytes, stem cells of dental apical papilla, and mouse melanoblastic progenitor cells [50,51,52,53,54]. More recently, we demonstrate that piggyBac transposon-mediated stable expression of SV40 T antigen is a more efficient approach to the immortalizing primary cells [60]. The most noteworthy features shared by all of the SV40 T antigen-immortalized cells include that 1) they are reversible and become mortal upon the removal of SV40 T antigen from the immortalized cells; 2) stable expression of SV40 T antigen does not completely inhibit the differentiation potential although the removal of SV40 T antigen has been shown to improve the terminal differentiation; and 3) the immortalized cells are non-tumorigenic in immune compromised mice, even after an extended observation period [50,51,52,54].…”

“…The coding regions of Cre recombinase and firefly luciferase (FLuc) were PCR amplified and subcloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in our recently engineered packaging line 293pTP [57], resulting in adenoviruses Ad-Cre and Ad-FLuc, which also express GFP as a marker for monitoring infection efficiency. Analogous adenovirus only expressing GFP (Ad-GFP) was used as a control [44,58,59,60,61,62,63,64,65,66]. For all adenovirus infections, polybrene (8µg/ml) was added to the culture medium in order to enhance transgene transduction efficiency [67].…”

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs)

“…The ALP activities were assessed using the modified Great Escape SEAP chemiluminescence assay (BD Clon-tech) and/or histochemical staining as described previously [31, 32, 63, 64]. Early osteogenic marker ALP activity was assessed at 3, 5, and 7 days after adenovirus infection.…”

Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses

“…The iMEF cells are mouse mesenchymal stem cells (MSCs) previously described [17, 18]. These cells lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C in 5% CO 2 as described [19, 20].…”

“…293T expressing single genes, the M series, and the RAPA line were accomplished by co-transfection of single or combined pBC2 expression vectors with a piggyBac transposase expression vector into 293T cells with Lipofectamine (Invitrogen, Grand Island, NY, USA), as described [18]. Stable cells were selected in the presence of Blasticidin S (at the final concentration of 5µg/ml) for 5-7 days with repeated plating.…”