Browsing by Subject "molecular biology"

Now showing 1 - 11 of 11

Adventures in Sequencing
(The Texas Scientist, 2019) The Texas Scientist
Dan Leahy
(The Texas Scientist, 2018) The Texas Scientist
Erasing Birth Defects
(The Texas Scientist, 2016) The Texas Scientist
Generating lightly immortalizing T-Cells to study the host immune response and immunotherapies
(2023-04) Hoffman, Ashley S.; Umlauf, Benjamin J.
Immortalized T-cell lines are key tools for studying disease response, immune pathways, and novel treatments. However, many of these lines are transformed into undefined cancerous cells to maintain unlimited growth potential, limiting their utility as an accurate model of host T-cells. This creates a need for T-cell lines that mimic healthy behavior, especially for testing T-cell-mediated therapies (e.g., CAR T-cell and checkpoint inhibitor) and T-cell exhaustion, a phenomenon observed in patients undergoing intense chronic therapies, that can still be shared amongst the scientific community. To this end, we have developed a method of lightly immortalizing T-cells from human donor peripheral blood monocytes (PBMCs), without producing a cancerous phenotype, by only expressing 2 genes: human telomerase reverse transcriptase (hTERT) and cyclin-dependent kinase 4 (Cdk4). Cdk4 and hTERT are key genes in driving cell cycle and survival. Over expression of these genes should create a slowly dividing T-cell population that is still capable of responding to immune cues and signals. Lightly immortalized T-cells will serve as a key resource for studying T-cell-mediated therapies such as chimeric antigen receptor (CAR) T-cells and effects of chronic treatment, including T-cell exhaustion. Here, we present a robust method for lightly immortalizing T-cells from PBMCs. Our protocol successfully extends the lifespan of PBMCs and selects for effector and helper T-cell populations in two donor PBMCs. To verify successful transfection and selection of T-cells hTERT and Cdk4 we used western blotting and FACs. We also observed typical T-cell behavior post-immortalization and achieved a resting population of healthy, actively dividing T-cells with measuring expression of CD3. In conclusion, this method provides a straightforward, reproducible, and shareable tool for understanding T-cell response to treatment and studying T-cell exhaustion in multiple patients.
Hook 'Em Health
(The Texas Scientist, 2019) The Texas Scientist
Humanizing Yeast
(The Texas Scientist, 2016) The Texas Scientist
Mu-Jie Lu
(The Texas Scientist, 2020) The Texas Scientist
Optimization of chromatin immunoprecipitation
(2012) Shah, Ronak; de Lozanne, Arturo
Transcription factors play a vital role in controlling cell growth, and locations of their binding sites at various points of the cell cycle can provide clues about malfunctions in eukaryotic growth, such as cancer. Our research focuses on transcriptional regulation of the eukaryotic cell cycle, using Saccharomyces cerevisiae as the model organism. We are observing binding patterns of affinity-tagged MCM1, SWI4, SWI5, FKH1, FKH2, and ACE2 transcription factors. These binding sites are discovered and isolated in vivo using chromatin immunoprecipitation (ChIP) followed by high-throughput, next-generation sequencing to map them to the genome for further analysis. The procedure locks transcription factors to their binding sites on DNA, and then eliminates the extraneous DNA to isolate the genes of binding site alone. However, ChIP generates a relatively low yield of DNA, often contaminated, and our research focuses on optimizing elements of the protocol to produce a higher, purer, output. Optimized lysis methods have reduced time and increased output of DNA, and sonication cycles have been adjusted to yield a more uniformly sheared mixture of DNA. The efficacy of sonication is evaluated through diagnostic gel electrophoresis and interpretation of visual results. In addition, the impact of pre-clearing on clarity of final yields was studied. DNA output and purity is repeatedly tested at various breakpoints of the ChIP procedure to ensure that the optimization modifications are delivering higher yields. DNA purity is tested using polymerase chain reaction (PCR) to find previously characterized target regions in the genome along with positive and negative controls to ensure homogeneity of the sample. Thus, by optimizing ChIP, we can obtain a highly accurate DNA sample more suitable for next-generation sequencing.
Optimizing the production of and engineering polyketide synthases
(2019-05-09) Shah, Prachi; Keatinge-Clay, Adrian
Polyketide synthases are enormous modular enzymes that synthesize large acyl chains known as polyketides. Common polyketides include pharmaceuticals such as antibiotics, immunosuppressants, and antifungals1. The mechanism of polyketide synthases is analogous to an assembly line. A group of enzymes work together to extend and process an acyl intermediate, forming new carbon-carbon bonds and setting stereocenters. The group of enzymes, a “module”, consists of a set of processing enzymes on its N-terminal end, an acyl carrier protein in its center, and a ketosynthase at its C-terminal end2. Understanding the chemical machinery of and engineering polyketide synthases is useful for future pharmaceutical applications. This work focuses on creating and optimizing the synthesis of two polyketides: venemycin and the first three modules of erythromycin. Erythromycin triketide lactone was produced in vitro and in vivo, and engineering experiments were attempted. Venemycin was produced in vitro, and its production was optimized by modifying various parameters including pH and buffer concentrations.
Silencing TFEB Suppresses Mammary Gland Acini Development and Proliferation
(2023-04) Goyal, Ria; Van Den Berg, Carla
Breast cancer is the most common type of cancer in the world. The aggressive and invasive nature of these tumors is explicable by the presence of cancer stem cells, which have self-renewal and initiation potentials that are analogous to the capabilities seen in normal mammary stem cells. The characterization of a protein’s role in a normal mammary gland, along with how this function is altered in cancerous tissue, enables enhanced understanding of the potential for specific novel drug targets. Transcription factor EB (TFEB) is currently known for its role as a master lysosomal regulator, and it has been shown to assist with cancerous progression through metabolic reprogramming. Yet, the key functions served by TFEB in mammary acini formation and its effects on mammary stem cells have never been determined. We have shown that the silencing of TFEB interrupts and disorganizes the development of mammary acini, unlike mere transcriptional inactivation. Furthermore, TFEB was not found to directly regulate the differentiation of mammary stem cells into a luminal lineage. Instead, TFEB appears to markedly contribute to the early proliferation of MCF10A cells, but additional investigation is necessary to characterize the mechanism. This work will guide future exploration into TFEB's potential as a drug target.
Silencing TFEB Suppresses Mammary Gland Acini Development and Proliferation
(2023-05) Goyal, Ria
Breast cancer is the most common type of cancer in the world. The aggressive and invasive nature of these tumors is explicable by the presence of cancer stem cells, which have self-renewal and initiation potentials that are analogous to the capabilities seen in normal mammary stem cells. The characterization of a protein’s role in a normal mammary gland, along with how this function is altered in cancerous tissue, enables enhanced understanding of the potential for specific novel drug targets. Transcription factor EB (TFEB) is currently known for its role as a master lysosomal regulator, and it has been shown to assist with cancerous progression through metabolic reprogramming. Yet, the key functions served by TFEB in mammary acini formation and its effects on mammary stem cells have never been determined. I have shown that the silencing of TFEB interrupts and disorganizes the development of mammary acini, unlike mere transcriptional inactivation. Furthermore, TFEB was not found to directly regulate the differentiation of mammary stem cells into a luminal lineage. Instead, TFEB appears to markedly contribute to the early proliferation of MCF10A cells, but additional investigation is necessary to characterize the mechanism. This work will guide future exploration into TFEB's potential as a drug target.