Quantitative Characterization of Conserved Noncoding Regions by Flow Cytometry

<p>Spatial and temporal regulation of developmental gene transcription often involves regulatory sequences found in noncoding DNA separate from the gene’s promoter. Conservation of a noncoding region (CNR) across divergent species may indicate a regulatory region critical to basic morphogenesis. CNR-green fluorescent protein (GFP) constructs can demonstrate spatial and temporal CNR activity by microscopy visualization. However, characterization of regulatory modules within a CNR requires quantitative, in addition to qualitative, analysis. The study presented here describes the development and implementation of a novel application of flow cytometry in the quantitative characterization of CNRs. The technique couples fluorescent microscopy localization with flow cytometry distribution and expression level analysis, creating a technique more powerful than either on its own. To optimize the flow cytometric analysis technique, we examined the limb-specific Shh regulatory region, LSSRR. LSSRR is a CNR that tightly regulates the specific spatiotemporal expression of Shh, a crucial morphogen for limb patterning. Our data demonstrate that flow cytometric analysis can identify and quantify changes in the distribution and intensity of CNR activity. Thus, this technique can be used to characterize conserved regulatory modules necessary for limb morphogenesis. However, it is likely that other organs and tissues utilize specific CNRs. The technique developed here is widely applicable outside of limb development. This enhances our ability to characterize CNR-related regulatory modules in general, and the technique can be used to advance knowledge of organ patterning during development.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Basic Sciences

Year

2014

Author dc:creator

• Brown, Amber

Contributors dc:contributor

• Oberg, Kerby C.
• Langridge, William
• Payne, Kimberly J.

• Biochemistry
• Biochemistry, Biophysics, and Structural Biology
• Medicine and Health Sciences
• Flow Cytometry; Gene Expression Profiling; Reverse Transcriptase Polymerase Chain Reaction; Fibroblast Growth Factors; Receptor Protein-Tyrosine Kinases; Receptors - Retinoic Acid; Mesoderm; Electroporation; Limb buds; Models - Biological
• Development Gene Transcription; Noncoding DNA; Morphogenesis; CNR-Green flourescent protein; Fluorescent Microscopy Localization;

Statement dc:rights

• This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/200

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-1199