Truncating mutations in the gene ASXL1 cause a rare genetic condition called Bohring-Opitz Syndrome (BOS); however, the exact mechanisms and the role of ASXL1 in this neurodevelopmental disorder remain elusive. Furthermore, all commercially available antibodies for ASXL1 do not reliably detect endogenous ASXL1, making many of the experiments that are needed to characterize its function impossible. To address this gap, we have developed an induced pluripotent stem cell (iPSC) line that has homozygous edits at the C-term of endogenous ASXL1 with the self-labeling Halo tag. We will characterize the edited iPSCs and the edited Neural Progenitor Cells (NPCs) derived from these iPSCs. We will confirm the edit using Sanger Sequencing and the cell type using various markers and performing immunofluorescent assays (IFAs). In order to capitulate the expression of ASXL1 and the reliability of the Halo tag, we will also use a Halo antibody to detect ASXL1-Halo at the protein level using Western Blot Analysis and IFAs. We expect to finally be able to detect endogenous ASXL1 using the Halo tag at least at the NPC stage, if not also at the iPSC state, as expression of ASXL1 is likely higher in the more relevant NPC cell type. The characterization of these edited lines is crucial to establish the efficacy of this cellular model in eventually studying the effect of truncating mutations on early neurodevelopment as well as unraveling the exact mechanisms through which mutations in ASXL1 cause BOS.

This project contributes to the foundational goal of functional genomics by identifying and attempting to clone a previously uncharacterized gene. Using BLAST, Echinobase, and InterPro, the gene was determined to be the Trafficking Protein Particle Complex Subunit 12 (TRAPPC12) in the Strongylocentrotus purpuratus species. TRAPPC12 is part of the TRAPP complex, which helps transport vesicles from the endoplasmic reticulum to the Golgi apparatus. A phylogenetic analysis revealed significant conservation of TRAPPC12, making it a particularly valuable target for further investigation. The gene was amplified via PCR, and the product was verified by gel electrophoresis and then purified, which was confirmed using Nanodrop spectrophotometry. The gene was then ligated into a pGEM-T vector and transformed into prepared DH5α competent cells. Blue-white colony screening showed the cells were competent, and white colonies suggested the transformation was successful; however, colony PCR did not confirm successful ligation. Bacterial cultures were inoculated, and plasmid DNA was extracted. A restriction digest followed by gel electrophoresis is required to confirm the presence of the insert and validate cloning success. While the gene's identity was confirmed, the success of its cloning remains to be determined. Confirming successful cloning will enable future studies of TRAPPC12 expression, regulation, and its potential role in intracellular trafficking.

ATP-binding cassette transporters utilize ATP hydrolysis to facilitate the transport of molecules across membranes. Specifically, ABCE1, a member of the ABC protein family, was found to be involved in translational regulation and ribonuclease inhibition. To generate a reliable source of this gene for future research projects, our primary aim was to identify and clone the ABCE1 gene using standard bioinformatic and molecular biology techniques. BLAST, InterPro, and Echinobase were used to identify the ABCE1 gene. To further confirm the identity of the gene, a phylogenetic tree of related paralogs and orthologs was generated. The gene was then amplified via PCR, utilizing agarose gel electrophoresis and silica column purification for size and purity confirmation. The subsequent gene DNA was ligated into a plasmid vector and propagated in bacteria. The resulting clones were verified through colony PCR, restriction digest analysis, and Sanger sequencing. The final Sanger sequencing definitively confirmed the identity of the cloned insert, successfully providing a readily available source of the ABCE1 gene to explore its precise role in ribosomal recycling mechanisms in the Pacific purple sea urchin.

S. purpuratas, a species of sea urchin commonly known as the purple sea urchin, can be found across the eastern edge of the Pacific Ocean. It is a key organismal model for understanding developmental biology and providing evolutionary insights into differences between invertebrates and vertebrates. This project aimed to explore a single gene within the species, identify its function and family, and clone it. The gene identity was determined utilizing databases such as BLAST, Echinobase, and Interpro, yielding the gene name, PGA55. This gene is a GPI-anchored adhesin-like protein involved in cell adhesion and signal transduction. It has various other roles due to its homology as a glycoprotein in different organisms, which was determined via phylogenetic analysis. Following identification, the gene was amplified by PCR. Amplification was verified via gel electrophoresis and followed by silica column chromatography. The isolated DNA was ligated to a plasmid vector and transformed into highly competent E. coli; transformation was analyzed via Colony PCR and restriction enzyme digest. Transformation efficiency and ligation efficiency were both successfully calculated. Colony PCR was unsuccessful, and the restriction digest was also unsuccessful in verifying the success of the cloning. Future goals of the project could have been functional experimentation, such as an in situ hybridization or a conditional Cre-Lox knockout.

Neurodegenerative retinal diseases affect over 300 million people worldwide, causing irreversible blindness. These diseases can be classified into age-related macular degeneration, diabetic retinopathy, and glaucoma. Extensive genetic heterogeneity, due to various mutation, is a big obstacle in developing gene therapy. There are 4 approaches to gene therapies: gene editing, gene silencing, gene replacement, and modifier gene therapy. I will be focusing on Adeno-associated virus as the gene therapy. AAVs are useful in delivering elements to specific cell types. Studies have shown that cis-regulatory modules, such as promoters and enhancers specific to cell types, are able to guide specific delivery of AAVs. We focus on identifying cell-type-specific genes, particularly foveal-enriched genes. Studies have identified numerous differentially expressed genes in various retinal cell types across species such as humans, macaques, marmosets, and mice. We have obtained chromatin accessibility data from ATAC-seq assays for these genes, from which CRM peaks can be identified. We identified 51 candidate CRMs and certain guide cell-specific expressions. I aim to validate whether the CRMs can guide AAV to target specific retinal neurons in mice using molecular cloning to insert the CRMs into AAV plasmids, along with intravitreal injections into mouse eyes. Immunohistochemistry and confocal imaging will be used to validate expression of reporter markers. The project will contribute to creating a toolbox for targeting specific cells.