Steven Hansberry

Discovery Chemistry

As an SRA, my primary project to create isogenic disease models while acting as a team member to coordinate and assist in secondary projects. Creation of iPSC isogenic disease models: • Optimization of selection methods to enrich and isolate correctly edited iPSCs • Optimization of CRISPR Ribonuclear protein and plasmid transfection efficiencies • Pioneering transient antibiotic selection • Maintenance of iPSC lines 3D culture collaboration • Coordination of sample transfer • Creation of 3D constructs from mouse bone marrow • RNA extraction and qPCR Other Projects • Targeted mutagenesis of plasmid vectors • Mouse osteoblast extractions

As a CIRM intern and master’s student, I aimed to understand the mechanisms behind a severe form of Fibrodysplasia Ossificans Progressiva by using CRISPR to introduce the disease-causing point mutation into wild-type cell lines. Alongside this, I drove forward a secondary project to establish a protocol for 3D culture of murine bone marrow. CRISPR editing to create disease models: • Designed and tested multiple CRISPR systems to select for high editing efficiencies • Created and optimized a digital droplet PCR system to quantify editing efficiencies • Designed restriction fragment analysis as a secondary mutant detection method • Optimized selection methods to enrich mutant populations in iPS and HEK293T cells • Isolated a correctly edited HEK293T cell line indicating expected differences in signaling 3D culture collaboration • Tested cell density, timelines, and media conditions to yield a protocol for a pilot experiment • Preformed 3D culture for pilot experiments • Troubleshot RNA extraction and staining • Coordinated between institutions Presentations and Writing • Presented at lab and project meetings • Created and presented a poster • Wrote and defended a master’s thesis

PI: Mark Chan Ph.D, Department of Biology San Francisco State University I was part of a team which investigates organelle size homeostasis, a process relevant to all cellular life. To understand this we studied budding yeast during cell division, specifically the transfer of mother vacuole to daughter cell. I contributed to this work by staining and imaging yeast vacuole over time during cell division. By quantifying these images we can measure the amount of Skills Microscopy: Confocal 3D imaging - Zeiss 710 Image j: Brightness measurement, Macros Yeast Culture and staining