Lecture 1 – Genetic Manipulation Flashcards
Teratomas: General Characteristics
Origin: Arises from germ cells forming eggs (oocytes) in females and sperm in males.
Age Occurrence: Typically found in young people.
Differentiation: Can differentiate into various cell types and tissues.
Composition: Contains pluripotent stem cells.
Types of Teratomas
Boys: Sacrococcygeal teratoma (often malignant).
Girls: Ovarian teratoma (often benign) with fully differentiated tissues.
Always Malignant: Testicular teratocarcinoma in boys.
Giant Cell Tumors (GCTs)
Group Membership: Teratomas belong to the category of giant cell tumors.
Pluripotent Origin: Arises from a pluripotent germ cell undergoing somatic differentiation.
Somatic Differentiation
Definition: Permanent change in gene expression.
Consequence: Descendant cells cannot fully develop into the organism.
Embryonal Carcinoma (EC) Cells
Culture Potential: Sacrococcygeal teratoma sample can be kept as EC cells.
Characteristics: Pluripotent and rarely totipotent.
Testicular Teratocarcinoma
Culture as EC Cells: Malignant cells can be kept in tissue culture.
Developmental Origin: Likely arises from defects in germ cell development in utero.
Terminology: Teratoma vs. Teratocarcinoma
Teratoma: Tumor with differentiated elements from all three germ layers.
Teratocarcinoma: Malignant tumors with EC cells, presumed malignant stem cells.
Demonstration of Cancerous Stem Cells
Experiment: Transplantation of a single EC cell resulted in a differentiated tumor.
Implication: EC cells are malignant, capable of self-renewal and differentiation.
Embryonal Carcinoma (EC) Cells and ES Cells
Comparison: Mouse EC cells thought comparable to inner cell mass (ICM) cells.
Isolation: ES cells isolated directly from the ICM for genetic modification.
Signaling Pathways and Early Development
Insight Source: Analysis of pathways regulating ES cell self-renewal/differentiation.
Applications: Understanding early embryonic development, cancer, and potential regenerative medicines.
Role of Retinoic Acid in Patterning
Evidence: Pluripotent EC cells show retinoic acid impact on HOX genes.
Importance: Retinoic acid required for normal patterning in vertebrates.
Pluripotent Stem Cells in Regenerative Medicine
Pioneering Attempt: Transplantation of NTERA2 EC cell-derived neurons into stroke patients.
Implication: Represents the initial use of pluripotent stem cells in regenerative medicine.
Key Transcription Factors for Pluripotency
Factors: OCT4, SOX2, NANOG.
Role: Maintain pluripotency in mouse and human EC and ES cells.
Potential of ES Cells for Tissue Replacement
Application: Surgical replacement of damaged tissues with ES cell-derived cells.
Examples: Oligodendrocytes for spinal cord injuries, treatments for diseases like macular degeneration.
EC Cells as In Vitro Models
Purpose: Used as in vitro models for early mouse development.
Limitations: Harbor genetic mutations, abnormal karyotypes.
Embryonic Stem Cells (ES Cells)
Definition: Pluripotent stem cells from the ICM of blastocysts.
Characteristics: Derived from preimplantation embryos, normal karyotypes, high telomerase activity.
Milestone in ES Cell Research (1998)
Researcher: James Thomson et al.
Achievement: Extraction and nurturing of human blastocyst-derived pluripotent cell lines.
Features: Normal characteristics, cultured for 4-5 months, able to differentiate into multiple tissues.
Applications and Future Prospects of ES Cells
Benefits: Valuable for human developmental biology, drug discovery, transplantation medicine.
Potential: Contribution to the development of all tissues in the organism.
Blastocyst and Inner Cell Mass (ICM)
Origin: Early conceptus forms a blastocyst.
Source: ICM is the origin of the entire embryo.
Histochemistry in Blastocyst Analysis
Markers: CDX2 in trophectoderm, OCT4 in ICM.
Method: Utilizes unique markers in different tissues for analysis.
Derivation and Maintenance of ES Cells
Process: Derived by dissociating the blastocyst and culturing the ICM.
Maintenance: ES cell lines can be maintained almost indefinitely under appropriate conditions.
Leukemia Inhibitory Factor (LIF) in ES Cells
Function: Prevents differentiation, allowing continuous cloning.
Result: ES cells form teratomas if introduced into mice, contribute to all parts of embryo development when reintroduced to the blastocyst.
Role of Key Transcription Factors
OCT4: Required for ICM cells, vital for pluripotency.
NANOG: Null cells lose pluripotency, develop as extra-embryonic tissues.
Signal Transduction and Pluripotency Maintenance
LIF Mechanism: Works through signal transduction to maintain NANOG, OCT4, SOX2, and Kif4 (pluripotency genes).
BMPs: Also maintain pluripotency gene levels to prevent cell differentiation.
ES Cell Manipulation through Electroporation
Procedure: Cells manipulated through electroporation.
Process: Cells placed in media with DNA solution, electric shock introduces surrounding DNA into the cell, inspected and selected cells isolated.
Application of ES Cells for Genetic Modification
Purpose: Used to create genetically modified mice.
Technique: ES cells are modified through a specific technique.
Introduction of Modified ES Cells
Process: Surviving ES cells capable of cloning are introduced into the host ICM blastocyst.
Host ICM Blastocyst: Implanted into the uterus of a pseudo-pregnant mouse.
Pseudo-Pregnant Mouse and Uterine Preparation
Condition: Mouse is pseudo-pregnant (swelling of uterine wall, vascularization) from a vasectomized mouse.
Purpose: Provides an environment for blastocyst implantation.
Birth of Chimeric Mice
Outcome: Female gives birth to a litter of variably chimeric mice.
Chimeric Offspring Identification: Usually, host blastocyst and donor ES cells are from different mouse strains with distinct coat colors.
Mate Chimeric Offspring with Wild Mice
Objective: Mate chimeric offspring with wild mice.
Result: Continue mating until a fully pigmented mouse appears.
Identification of Chimeric Offspring
Method: Chimeric offspring from different mouse strains have varying coat colors.
Purpose: Facilitates easy identification of chimeric mice.
Advantages of Using Different Mouse Strains
Strategy: Host blastocyst and donor ES cells from different strains.
Reason: Enhances visibility and recognition of chimeric offspring.
Completion of Pigmented Mice Generation
Process: Mating continues until a fully pigmented mouse is obtained.
Significance: Indicates successful integration of modified ES cells in the mouse lineage.
Utilization of Chimeric Mice
Applications: Study of gene function, disease modeling, and various experimental investigations.
Versatility: Chimeric mice serve as valuable tools in genetic research.
