Honors College Thesis


Building a cassette-based system for combined eukaryotic gene expression Public Deposited

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  • Gene regulatory networks control the state of a cell. Models of such networks have been produced for sea urchin development and suggest that cell type may be defined by distinct network kernels. Such kernels are composed of a set of sequence specific DNA-binding transcription factors (SSTFs) that maintain each other’s expression through cell type-specific cis-regulatory modules. Ectopic expression of some individual SSTFs leads to trans-differentiation, or the conversion of one cell type to another. Our hypothesis is that trans-differentiation will be more efficient and targeted if specific SSTF combinations, corresponding to those of a network kernel, are expressed ectopically. A transient, polycistronic, cassette-based expression system was designed and built. This system allow for Lbx1-dependent network kernels to be transiently introduced into heterologous cells. Transfecting a cell with a specific combination of SSTFs may trigger establishment of the corresponding endogenous network kernel and thereby lead to trans-differentiation. Five SSTF ORFs (Lbx1, Lmx1b, Pax2, Pax8, and Isl1) were PCR amplified with gene-specific primers that included AsiSI and PacI restriction sites. A base bacterial vector was created with a custom polylinker to receive the PCR-generated minimal ORFs. This custom polylinker was constructed so that PCR-generated minimal ORFs could readily be transferred to other vector systems by classic recombinant methods. The AsiSI/PacI minimal ORF fragments are transferred to a pcDNA 575/576 mammalian expression vector containing the IRES ORF cassette to generate IRES-ORF or ORF-IRES expression cassettes. Polycistronic expression cartridges of fluorescent proteins and SSTF kernels need to be completed before functional tests can be done.
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