Congenital muscular dystrophies (CMD) are a group of rare genetic disorders that are diagnosed postnatally, cause progressive weakness and paralysis in infants and toddlers, and currently have no specific treatment or cure. LAMA2-related congenital muscular dystrophy is caused by the loss of a critical protein, laminin-211, that provides structural support of muscle fibers by connecting the muscle membrane to the extracellular matrix. In this study we used patient-derived induced pluripotent stem cells (iPSCs) to develop an in vitro model of the disease. We differentiated iPSCs of a parent and her unaffected parent (carrier) control into skeletal muscle progenitor cells (SMPCs) and subsequently into contractile myotubes. We observed that the LAMA2- iPSCs form less robust neural-crest-like clusters during directed differentiation to SMPCs, suggesting a potential role of the supporting cell population. Further differentiation into myotubes demonstrated differences in cellular fusion and myotube organization in LAMA2-deficient cells. Our studies show stem cell-based in vitro modeling of LAMA2-related congenital muscular dystrophy can be used to serve as a patient-specific platform to better understand the processes of this disease and help advance therapeutics.

Transcription Factor 12 (TF12) belongs to the neuronal differentiation E-box binding family and plays a critical role in neural development through its basic helix-loop-helix and finulus domains. This domain enables dimerization and binding to E-box DNA sequences, activating gene expression essential for neural lineage specification. However, the specific role of TF12 in Strongylocentrotus purpuratus development remains largely unexplored. This study aimed to identify an unknown gene suspected to be TF12 and determine whether it could be successfully cloned into a plasmid. The unknown gene was identified using bioinformatics tools such as BLASTn, BLASTx, Echinobase, and InterPro. PCR was used to amplify the target gene, which was subsequently ligated into a plasmid vector and transformed into competent bacteria. The presence of white colonies showed successful transformation. These results confirmed that the gene of interest was indeed TF12 and was successfully cloned, though further corroboration such as DNA sequencing is needed. A phylogenetic analysis was performed to assess the genetic relationships and evolutionary context of TF12. Overall, this study displays the value of PCR in gene cloning and highlights the importance of downstream verification steps, while demonstrating the value of phylogenetic analysis in identifying conserved functional elements of TF12 that could inform future functional studies on its role in sea urchin neurodevelopment.

The Wnt signaling pathway plays a critical role in cell proliferation, differentiation, and development. The aim of this study was to identify and clone an unknown coding region of an unknown sea urchin gene. BLAST, InterPro, and phylogenetic analyses identified and confirmed the unknown gene to be Frizzled Class Receptor 9 (FZD9), a member of the Frizzled family of G protein-coupled receptors involved in Wnt signaling. The unknown gene sequence was amplified using PCR with designed primers, its products were then purified and ligated into plasmid vectors. Subsequent bacterial transformation, plasmid extraction, and restriction digest results showed inconclusive ligation and cloning of FZD9. Future work will focus on optimizing plasmid redigestion and religation of FZD9, followed by sequencing to confirm its integration. This study enhances our understanding of sea urchin genetics, further contributing to ongoing efforts to characterize genes and their respective functions involved within the Wnt signaling pathway.

An unknown gene from Strongylocentrotus purpuratus was identified and characterized with the goal of cloning a segment of its coding region into a plasmid. A BLAST analysis identified the unknown sequence as the forkhead box G1 (foxg1) gene. Further characterization was carried out using Echinobase, InterPro, and phylogenetic analysis. Foxg1 encodes a transcription factor important in regulating embryonic development, specifically in cell fate specification and cell differentiation. It is a member of the forkhead/fox family. After amplifying the target region of foxg1 using PCR, the DNA inserts were ligated into PGEMT vectors and then transformed into competent cells. The success of the cloning was assessed using colony PCR analysis, restriction digest analysis, and DNA sequencing. While the colony PCR and restriction digest analysis indicated the presence of the correct DNA insert in the vector, the DNA sequencing analysis revealed that foxg1 was not inserted into the vector. Instead, the zinc finger E-box binding homeobox 2 (zeb2) sequence was found in the plasmids. As a result, the initial project goal of cloning the target unknown gene into a plasmid was not achieved. The foxg1 gene was not correctly cloned. The experimental process needs to be repeated to correctly isolate and clone the foxg1 gene. Once successful, knockout experiments can be performed to further characterize the gene’s role in embryonic development.

Mitochondrial DNA (mtDNA) encodes essential components of oxidative phosphorylation and plays a critical role in cellular energy metabolism. In domestic dogs (Canis lupus familiaris), mtDNA variation has been associated with breed-specific traits and heritable diseases, offering a valuable model for studying mitochondrial genetics. This project aims to develop a computational pipeline to identify genetic variants in canine mtDNA, focusing on single nucleotide polymorphisms (SNPs) and structural variants. The pipeline includes steps for quality control, read trimming, alignment to a reference genome, duplicate marking, and base quality recalibration. Variant calling and downstream analyses were used to assess patterns of mitochondrial variation across individuals. By characterizing mtDNA in dogs, this study contributes to the fields of veterinary genetics and comparative genomics, with potential insights into the evolutionary conservation of mitochondrial function and relevance to human health.