Lecture 1 – Genetic Manipulation

Question 1

Teratomas: General Characteristics

Answer 1

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.

Question 2

Types of Teratomas

Answer 2

Boys: Sacrococcygeal teratoma (often malignant).
Girls: Ovarian teratoma (often benign) with fully differentiated tissues.
Always Malignant: Testicular teratocarcinoma in boys.

Question 3

Giant Cell Tumors (GCTs)

Answer 3

Group Membership: Teratomas belong to the category of giant cell tumors.
Pluripotent Origin: Arises from a pluripotent germ cell undergoing somatic differentiation.

Question 4

Somatic Differentiation

Answer 4

Definition: Permanent change in gene expression.
Consequence: Descendant cells cannot fully develop into the organism.

Question 5

Embryonal Carcinoma (EC) Cells

Answer 5

Culture Potential: Sacrococcygeal teratoma sample can be kept as EC cells.
Characteristics: Pluripotent and rarely totipotent.

Question 6

Testicular Teratocarcinoma

Answer 6

Culture as EC Cells: Malignant cells can be kept in tissue culture.
Developmental Origin: Likely arises from defects in germ cell development in utero.

Question 7

Terminology: Teratoma vs. Teratocarcinoma

Answer 7

Teratoma: Tumor with differentiated elements from all three germ layers.
Teratocarcinoma: Malignant tumors with EC cells, presumed malignant stem cells.

Question 8

Demonstration of Cancerous Stem Cells

Answer 8

Experiment: Transplantation of a single EC cell resulted in a differentiated tumor.
Implication: EC cells are malignant, capable of self-renewal and differentiation.

Question 8

Embryonal Carcinoma (EC) Cells and ES Cells

Answer 8

Comparison: Mouse EC cells thought comparable to inner cell mass (ICM) cells.
Isolation: ES cells isolated directly from the ICM for genetic modification.

Question 9

Signaling Pathways and Early Development

Answer 9

Insight Source: Analysis of pathways regulating ES cell self-renewal/differentiation.
Applications: Understanding early embryonic development, cancer, and potential regenerative medicines.

Question 10

Role of Retinoic Acid in Patterning

Answer 10

Evidence: Pluripotent EC cells show retinoic acid impact on HOX genes.
Importance: Retinoic acid required for normal patterning in vertebrates.

Question 11

Pluripotent Stem Cells in Regenerative Medicine

Answer 11

Pioneering Attempt: Transplantation of NTERA2 EC cell-derived neurons into stroke patients.
Implication: Represents the initial use of pluripotent stem cells in regenerative medicine.

Question 12

Key Transcription Factors for Pluripotency

Answer 12

Factors: OCT4, SOX2, NANOG.
Role: Maintain pluripotency in mouse and human EC and ES cells.

Question 13

Potential of ES Cells for Tissue Replacement

Answer 13

Application: Surgical replacement of damaged tissues with ES cell-derived cells.
Examples: Oligodendrocytes for spinal cord injuries, treatments for diseases like macular degeneration.

Question 14

EC Cells as In Vitro Models

Answer 14

Purpose: Used as in vitro models for early mouse development.
Limitations: Harbor genetic mutations, abnormal karyotypes.

Question 15

Embryonic Stem Cells (ES Cells)

Answer 15

Definition: Pluripotent stem cells from the ICM of blastocysts.
Characteristics: Derived from preimplantation embryos, normal karyotypes, high telomerase activity.

Question 16

Milestone in ES Cell Research (1998)

Answer 16

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.

Question 17

Applications and Future Prospects of ES Cells

Answer 17

Benefits: Valuable for human developmental biology, drug discovery, transplantation medicine.
Potential: Contribution to the development of all tissues in the organism.

Question 18

Blastocyst and Inner Cell Mass (ICM)

Answer 18

Origin: Early conceptus forms a blastocyst.
Source: ICM is the origin of the entire embryo.

Question 19

Histochemistry in Blastocyst Analysis

Answer 19

Markers: CDX2 in trophectoderm, OCT4 in ICM.
Method: Utilizes unique markers in different tissues for analysis.

Question 20

Derivation and Maintenance of ES Cells

Answer 20

Process: Derived by dissociating the blastocyst and culturing the ICM.
Maintenance: ES cell lines can be maintained almost indefinitely under appropriate conditions.

Question 21

Leukemia Inhibitory Factor (LIF) in ES Cells

Answer 21

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.

Question 22

Role of Key Transcription Factors

Answer 22

OCT4: Required for ICM cells, vital for pluripotency.
NANOG: Null cells lose pluripotency, develop as extra-embryonic tissues.

Question 23

Signal Transduction and Pluripotency Maintenance

Answer 23

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.

Question 24

ES Cell Manipulation through Electroporation

Answer 24

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.

Question 25

Application of ES Cells for Genetic Modification

Answer 25

Purpose: Used to create genetically modified mice.
Technique: ES cells are modified through a specific technique.

Question 26

Introduction of Modified ES Cells

Answer 26

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.

Question 27

Pseudo-Pregnant Mouse and Uterine Preparation

Answer 27

Condition: Mouse is pseudo-pregnant (swelling of uterine wall, vascularization) from a vasectomized mouse.
Purpose: Provides an environment for blastocyst implantation.

Question 28

Birth of Chimeric Mice

Answer 28

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.

Question 29

Mate Chimeric Offspring with Wild Mice

Answer 29

Objective: Mate chimeric offspring with wild mice.
Result: Continue mating until a fully pigmented mouse appears.

Question 30

Identification of Chimeric Offspring

Answer 30

Method: Chimeric offspring from different mouse strains have varying coat colors.
Purpose: Facilitates easy identification of chimeric mice.

Question 31

Advantages of Using Different Mouse Strains

Answer 31

Strategy: Host blastocyst and donor ES cells from different strains.
Reason: Enhances visibility and recognition of chimeric offspring.

Question 32

Completion of Pigmented Mice Generation

Answer 32

Process: Mating continues until a fully pigmented mouse is obtained.
Significance: Indicates successful integration of modified ES cells in the mouse lineage.

Question 33

Utilization of Chimeric Mice

Answer 33

Applications: Study of gene function, disease modeling, and various experimental investigations.
Versatility: Chimeric mice serve as valuable tools in genetic research.