Lecture 17: Genetic Manipulation in ES Cells

Lecture 17: Genetic Manipulation in Embryonic Stem Cells and Animals

• Totipotency, pluripotency, and germline biology are discussed.
• Blastocyst development (days 3-5): Crucial video for understanding the process. (Link provided)
• Embryonic stem (ES) cells: Defined as cells derived from the inner cell mass of a blastocyst, capable of differentiating into all cell types in the body. They can be cultured and used to generate genetically modified animals.
• Genetically modified animal models: ES cells enable researchers to create animals with specific genetic alterations for studying various biological processes.
• CRISPR experiments in ES cells: Gene editing tools like CRISPR are used to modify the genome of ES cells.
• Introduction to mouse genetics: Mouse genetics is important in researching genetic manipulations (knockouts, knock-ins, transgenics).
• Embryonic stem cell features: ES cells have the capacity for self-renewal and differentiation.
• CRISPR-mediated genome editing in ES cells: Described as a technique where DNA sequences are precisely altered using CRISPR.
• CRISPR-mediated genome editing in creating genetically modified animal models: Explains how gene editing is used to create genetically modified animals.
• Knockout, knock-in, versus transgenic mice: Different genetic manipulation strategies that produce genetically altered mice are differentiated.
• Making pluripotent cells (iPS cells): Technique of inducing pluripotency by forcing expression of particular transcription factors.
• Totipotency: Ability of a single cell to develop into a complete adult organism. ES cells exhibit pluripotency, the capacity to differentiate into all cell types but not a whole organism.
• ES cell isolation and culture: Techniques used for isolating and maintaining human or mouse ES cells. Includes a layer of fibroblast feeder cells to support their growth.
• Chimera mice: Embryos formed by introducing genetically modified ES cells into the blastocyst of a wild-type mouse, resulting in animals with a mix of genetically modified and normal cells.
• Conventional gene targeting in mice: Using ES cells to create targeted alterations in the genome of mice.
• CRISPR experiments in ES cells: Involves similar strategies used in other cell types, with many applications in stem cell research (in vitro studies, differentiation, organoids, and therapies).
• Introduction to mouse genetics: Discusses different types of genetic modifications to specific genes, such as knockouts, knock-ins, and transgenics. Highlights how these techniques are used to create genetically modified mice.
• Viral infection/transfection: Methods for introducing DNA into ES cells (and other cells in similar experiments).
• Selectable markers: Used to identify and select cells with the desired genetic modification.
• Specific examples: The use of CRISPR and targeted gene editing in various applications are discussed including the H2AX gene.