The document describes developing a PiggyBac gene delivery system to generate autonomously luminescent stem cells. Conventional tracking methods for stem cells have limitations. The lux gene cassette allows autonomous bioluminescence without exogenous substrates or costly imaging. The study aims to develop a PiggyBac transposon system to stably transfect stem cells with the lux genes. Preliminary results show the lux genes were successfully cloned into a PiggyBac vector. Transfection of HEK293 cells showed no bioluminescent output yet stable antibiotic-resistant colonies were formed, requiring further troubleshooting of gene expression.

1. Developing a PiggyBac Gene Delivery System to Generate Autonomously
Luminescent Stem Cells
Winode Handagama1
, Gary Sayler2
1
Maryville College, Department of Chemistry; 2
The University of Tennessee, Knoxville, The Center for Environmental Biotechnology
Introduction
Stem cell-based therapies have shown to be effec-
tive in treating diverse types of injuries and dis-
eases. The means to actively quantify and track the
fate, survival and proliferation of stem cells post-
transplantation is crucial for successful treatment .
Conventional Methods
• MRI, MRS, SPECT, and PET imaging:
expensive machinery and technical
operating skills to acquire data.
• Fluorescent reporter molecules (i.e. GFP):
high levels of background fluorescence
in living tissue.
• Substrate-dependent bioluminescence imaging:
requires the addition of an exogenous
substrate.
The lux gene cassette encodes the enzymes and sub-
strates needed for autonomous bioluminescence, and
may be a cost-effective alternative to conventional
methods.
lux Gene Cassette
• autonomous bioluminescence without
exogenous substrate
• does not require costly imaging
devices
• no background bioluminescence in
mammalian tissues
Figure 1: A) Humanized lux gene cassette. B) lux genes en-
code enzymes and substrates for a constitutively bioluminescent
pathway.
Background
Figure 2: A) PiggyBac (PB) Transposon/Transposase system
allows for insertion of genes flanked by inverted terminal re-
peats (ITRs) from a PB vector into genomic DNA. B) Equine
Mesenchymal Stem Cells (EqMSCs) transiently expressing lux
genes.
Transient transfection of EqMSCs with the lux genes
was successful; this study’s aim is to develop a Pig-
gyBac Transposon delivery system to stably trans-
fect stem cells with the lux gene cassette.
Methods
Results
Figure 3: A restriction digest of the ligation product extracted
from E. coli with EcoRV (1), HindIII (2), and BamHI (3); the
ladder is on the far right (4).
The cloning of the lux genes into the PiggyBac Vec-
tor PB-Neo was successful.We proceeded to trans-
fect HEK 293 cells.
Figure 4: Lux genes cloned into a PiggyBac Vector, with ITR
sequences for Transposase delivery into mammalian cell lines.
Cells were imaged 24 hours post-transfection.
Figure 5: Bioluminescent output of HEK293 cells transfected
under 5 different conditions.The CMV positive controls indicate
the transfection system is working. There is no output from the
PB-EFLux-Neo transfections.
Conclusion
• The humanized lux gene cassette was successfully
cloned into a PiggyBac vector for transfection
into human cells: PB-EFLux-Neo.
• HEK293 Cells transfected with PB-EFLux-Neo
do not demonstrate a bioluminescent output 24
hours post-transfection. Although there was no
detectable bioluminescence, stable colonies were
formed after 2 weeks of G418, Geneticin®
selection.
Future Work
• Selection of colonies that display resistance to
G418, Geneticin®
.
• Troubleshoot construction of the plasmid for
hindrances to mammalian gene expression.
References
1 Xu, Tingting; Ripp, Steven; Sayler, Gary S. ;
Close, Dan M. (2014) Expression of a Humanized
Viral 2A-Mediated lux Operon Efficiently
enerates Autonomous Bioluminescence in Human
Cells. PLoS One 5: e96347
Acknowledgements
This work was funded through support from the National Sci-
ence Foundation (DBI-1156644), and would not have been pos-
sible without the continual guidance of Dr. Tingting Xu, Dr.
Steven Ripp, and Haylie Lam, as well as the the laboratory
facilities and instrumentation provided by the University of
Tennessee’s Center for Environmental Biotechnology. The au-
thors would also like to thank Dr. Steven Wilhelm and Dr.
Gary LeCleir for directing and organizing the REU program.