Browsing by Subject "Flow Cytometry"
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Item Beyond the cycle: investigating the sequencing, binding affinity, and utility of aptamers selected with CE-SELEX.(2023-02) Brinza, NicholasAptamers are single stranded DNA or RNA sequences that bind with high affinity to a target molecule. The discovery of sequences that can bind to a target of interest starts with a mixture of random sequences which are refined through repeated incubation with the target followed by amplification of binding sequences in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers produced by multiple SELEX rounds can assist bioanalytical scientific research through detecting molecules of interest in biosensors and have the potential to improve drug delivery to specific cells in humans. However, developing aptamers capable of useful application is difficult due to multiple unique challenges. Quantifying the ability for a pool of aptamers produced by SELEX to bind to a target has no single perfect method. In the field of aptamer research, scientists employ a variety of binding affinity tests which contain specific advantages and disadvantages. Reported values for binding affinity vary between different methods. When a pool does show evidence of most sequences binding, identifying the best sequences in a mixture of more than thousands of unique base pair arrangements provides a separate challenge. Finally, even after finding a sequence with evidence of strong binding to a desired target, there remains uncertainty if the aptamer will bind to the target as well in a real-world application as it does in a carefully controlled lab environment. This study examines aptamers at three distinct stages of development for separate capillary electrophoresis SELEX (CE-SELEX) experiments. For an aptamer already selected against transferrin receptor 1 (TfR1) with high affinity, a combination of flow cytometry and confocal microscopy provided evidence the aptamer did not significantly internalize into cancerous liver cells used to model human drug delivery. A separate aptamer pool, previously developed over 4 rounds of selection against low-density lipoprotein receptor (LDLR), is analyzed with preexisting bioinformatic tools as well as custom Python code on next generation sequencing (NGS) data, revealing a primer dimer sequence from polymerase chain reaction (PCR) which impacted the selection of aptamer length candidates but remained at a lower frequency than one possible aptamer candidate. After performing 4 rounds of CE-SELEX against mouse leptin, testing the binding affinity of rounds 1-3 with CE showed weak evidence of binding in early rounds which was lost in later rounds after correcting for an earlier false positive result. Documenting CE problems and optimization, even for an unsuccessful aptamer selection, may help improve future CE research, while detailing the experimental set up, discovery, and correction of the false positive result is important to help prevent future misleading conclusions.Item Duchenne muscular dystrophy and extraocular muscle: a potential sparing mechanism with therapeutic implications.(2009-10) Kallestad, Kristen MarieThis project investigates the role of extraocular muscle (EOM) progenitor cells in sparing the muscles from pathology associated with Duchenne Muscular Dystrophy (DMD). Mouse models of muscular dystrophy and wild type mice were analyzed by flow cytometry and cell culture for the size, heterogeneity and functional characteristics of stem and satellite cell populations of EOM and limb muscles. EOM have a 5-fold increase in progenitor cells compared with limb muscles. Additionally, an enriched population of cells expressing the stem cell marker CD34 but no other typical stem or differentiation markers (Sca-1, CD45, CD31, pax-7, m-cadherin) exists in the EOM. We refer to this population as EOMCD34 cells. The EOMCD34 cells are present in developing muscle, but only maintained in adult EOM, surviving in very aged animals. The EOMCD34 cells are also present in EOM of DMD model animals, but not their limb muscles. EOMCD34 cells are resistant to apoptosis and proliferate in vivo. Finally, these cells are capable of forming myotubes in vitro. The EOMCD34 cells may represent a primitive stem cell population, which is capable of maintaining life-long pools of myogenic precursor cells. Since EOM continuously remodel throughout life, unlike other skeletal muscle, it is logical that the proliferative potential of their precursor cells is enhanced. Since one proposed mechanism of DMD muscle destruction is exhaustion of the reparative progenitor cells, the EOMCD34 cells might prove useful for myoblast transplant therapies for DMD.