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<p>Synthetic biology facilitates both the design and fabrication of biological components
and systems that do not already exist in the natural world. From an engineering point
of view, synthetic biology is akin to building a complex machine by assembling simpler
parts. Complex genetic machines can also be built by a modular and rational assembly
of simpler biological parts. These biological machines can profoundly affect various
cellular processes including the transcriptional machinery. In this thesis I demonstrate
the utilization of biological parts according to synthetic biology principles to solve
three distinct transcription-level problems: 1) How to efficiently select for transgene
excision in induced pluripotent stem cells (iPSCs)? 2) How to eliminate transposase
expression following piggyBac-mediated transgenesis? 3) How to reprogram cell lineage
specification by the dCas9/gRNA transactivator-induced expression of endogenous transcription
factors? </p><p>Viral vectors remain the most efficient and popular in deriving induced
pluripotent stem cells (iPSCs). For translation, it is important to silence or remove
the reprogramming factors after induction of pluripotency. In the first study, we
design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming
elements in a single lentiviral vector expressed by a strong EF-1α promoter;
b) enables easy determination of lentiviral titer; c) enables transgene removal and
cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex
Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows
for transgene excision in a colony format. With our design, a reprogramming efficiency
comparable to that reported in the literature without boosting molecules can be consistently
obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy,
we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX)
in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase.
The robustness of this platform enables the implementation of an efficacious and cost-effective
protocol for iPSC generation and their subsequent transgenesis for downstream studies.</p><p>Transgene
insertion plays an important role in gene therapy and in biological studies. Transposon-based
systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism
have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon
is particularly promising due to its ability to integrate large transgenes with high
efficiency. However, prolonged expression of transposase can become a potential source
of genotoxic effects due to uncontrolled transposition of the integrated transgene
from one chromosomal locus to another. In the second study we propose a vector design
to decrease post-transposition expression of transposase and to eliminate the cells
that have residual transposase expression. We design a single plasmid construct that
combines the transposase and the transpositioning transgene element to share a single
polyA sequence for termination. Consequently, the transposase element is deactivated
after transposition. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk)
with the transposase. Therefore, cells having residual transposase expression can
be eliminated by the administration of ganciclovir. We demonstrate the utility of
this combination transposon system by integrating and expressing a model therapeutic
gene, human coagulation Factor IX, in HEK293T cells.</p><p>Genome editing by the efficient
CRISPR/Cas9 system shows tremendous promise with ease of customization and the capability
to multiplex distinguishing it from other such technologies. Endogenous gene activation
is another aspect of CRISPR/Cas9 technology particularly attractive for biotechnology
and medicine. However, the CRISPR/Cas9 technology for gene activation leaves much
room for improvement. In the final study of this thesis we show that the fusion of
two transactivation (VP64) domains to Cas9 dramatically enhances gene activation to
a level that is sufficient to achieve direct cell reprogramming. Targeted activation
of the endogenous Myod1 gene locus with this system leads to stable and sustained
reprogramming of mouse embryonic fibroblasts into skeletal myocytes. </p><p>In conclusion,
this dissertation demonstrates the power of utilizing biological parts in a rational
and systematic way to rectify problems associated with cell fate reprogramming and
transposon-based gene delivery. Through design of genetic constructs aided by synthetic
biology principles, I aspire to make contributions to the related fields of cellular
reprogramming, stem cell differentiation, genomics, epigenetics, cell-based disease
models, gene therapy, and regenerative medicine.</p>
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