Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Abstract

Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to the limited availability of piggyBac vectors with manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression.

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

“…293pTP cells were derived from HEK-293 cells and overexpression of human Ad5 pTP gene as previously characterized [50]. The cell lines are maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 mg/ml streptomycin, at 37°C in 5% CO 2 as described [51-53]. Unless indicated otherwise, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo-Fisher Scientific (Waltham, MA).…”

A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti-Proliferative and Anticancer Activities

“…All experimental procedures were carried out in accordance with the approved guidelines. Briefly, HeyA8 stably labeled with firefly luciferase (HeyA8-FLuc) was constructed with piggybac system [37,43,66]. Exponentially growing HeyA8-FLuc cells were infected with AdR-siIGF1R or Ad-RGFP for 36h and collected, resuspended at 10 7 cells/ml and injected subcutaneously into the flanks of athymic nude mice (Harlan Laboratories, 6-8 week old, male, 10 6 cells per injection, and 4-6 sites per mouse).…”

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

“…We have recently optimized the piggyBac transposon system, which has been shown to have significant advantages over retroviral vectors in making stable lines with high levels of transgene expression [18, 21, 22]. Using the PBC2 as a base vector we subcloned six genes of human adenovirus type 5 (i.e., E1A, E1B19K, E1B55K, pTP, DBP, and DNA Pol) and the host factor OCT1 into this vector (Fig.…”

“…In order to achieve a high level and stable transgene expression in 293 cells, we choose to use our optimized piggyBac transposon-based gene expression system [18, 21, 22], which can effectively integrate multiple copies of the transgene into relatively AT-rich regions of host chromosomes. We successfully established six types of genetically modified 293T M lines with different combinations of viral genes and/or OCT1 and found that the stable 293T M lines express high levels of transgenes.…”

“…A homemade piggyBac vector pBC2, which contains piggyBac terminal repeats (PB-TRs), core insulators (CIs) and the Blasticidin S selection marker, was used as the base vector [21, 22]. The coding regions of human Ad5 pTP, E1A, E1B19K, E1B55K, DBP, and DNA Pol were PCR amplified and/or assembled together from adenoviral genome-containing plasmid pJM17.…”