Lecture 2 - Mutagenesis and gene knockout in animals

Question 1

Ethical Concerns in Animal Research

Answer 1

Issue: Ethical concerns arise in the use of animals for genetic modification.
Concerns: Creation and maintenance of genetically modified mouse stocks, potential suffering, and unknown harmful effects.

Question 2

Regulation and Approval for Animal Research

Answer 2

Regulation: Highly regulated by the Animals (Scientific Procedures) Act 1986.
Approval: Projects involving animals require specific approval from the Home Office via a project license.
Competence: Individuals working with animals must possess a personal license, demonstrating competence.

Question 3

Cost-Benefit Analysis in Animal Research

Answer 3

Principle: Evaluates if the benefits of research outweigh the suffering of animals.
Criteria: Ethical justification based on the balance of sought-after results and potential animal suffering.

Question 4

Reduction, Refinement, and Replacement (3Rs)

Answer 4

Objective: Ethical approach to reduce the use of animals, refine experiments, and replace animals where possible with alternative models.
Application: Striving for more ethical practices in animal research.

Question 5

Genetic Manipulation for Human Health Understanding

Answer 5

Purpose: Understand genetic basis of human health and disease.
Methods: Over-expression, knockout, and control of gene expression.

Question 6

Creation of Designer Animals

Answer 6

Purpose: Genetic tagging or creation of ‘designer animals’ for research, medical, or important purposes.
Example: Disease models for studying conditions like cystic fibrosis (CFTR knockout).

Question 7

Spontaneous Mutations and Phenotype Changes

Answer 7

Occurrence: Spontaneous mutations may lead to observable changes.
Examples: PAX6 mutation results in small eyes, Vangl2 mutation leads to a looptail mouse, and Limk1 mutation causes clubfoot.

Question 8

Random Mutagenesis for Gene Exploration

Answer 8

Method: Exposing mice to mutagens (X-rays/chemical mutagens) to accelerate gene mutation rates.
Chemicals: ENU/EMS induce point mutations in the genome.

Question 9

Germ Line Mutations and Screening

Answer 9

Objective: Affect germ line of male mice, screening for dominant mutations after mating with wild-type female mice.
Outcome: Identification of interesting phenotypes, potential discovery of new genes related to diseases.

Question 10

Inbreeding for Homozygous Recessive Traits

Answer 10

Strategy: Inbreed mutant mice with wild-type to identify homozygous recessive traits.
Application: Uncover mutations in tissues, linking them to specific genes and diseases.

Question 11

Challenges in Animal Research

Answer 11

Issue: Large number of animals used, potential wastefulness in overlooking interesting mutations due to focused research areas.

Question 12

Gene Targeting (Knock-outs)

Answer 12

Objective: Delete genes from animals through homologous recombination in embryonic stem cells (ES cells).
Homologous Recombination: Occurs during meiosis where homologous sequences on maternal and paternal chromosomes align, cross over, and exchange genetic material.

Question 13

Targeting Vector or Transgene

Answer 13

Definition: DNA replacing the original DNA in homologous recombination.
Selection: Includes a neomycin resistance gene (neoR) for identification; can also include a green fluorescent protein gene.

Question 13

Introduction of New DNA into Cells

Answer 13

Method: Homologous recombination used to introduce new DNA into cells.
Frequency: Can occur accidentally at a low frequency in any cell at any time.

Question 14

Selection Process in Gene Targeting

Answer 14

Purpose: Identify cells that undergo homologous recombination.
Neomycin Resistance: Cells taking up the gene become resistant to neomycin; others die.
Additional Gene: Flanking sequences include a thymidine kinase gene targeted with antiviral drugs.

Question 15

Ensuring Correct Homologous Recombination

Answer 15

Strategy: Cells integrating the targeting vector survive neomycin but die due to the additional gene (TK) if incorrectly targeted.
Drug Sensitivity: Cells integrating TK become susceptible to antiviral drugs like ganciclovir.

Question 16

Verification of Integration

Answer 16

Process: PCR/Southern blotting performed on genomic DNA from survivors.
Check: Ensures the integrity of the integrated vector in successfully targeted ES cells.

Question 17

Implantation into Host Blastocysts

Answer 17

Procedure: Targeted ES cells injected into the inner cell mass (ICM) of host blastocysts.
Outcome: Chimeric offspring produced for further breeding.

Question 18

Low Efficiency of Homologous Recombination

Answer 18

Occurrence: Homologous recombination is rare, accounting for <2% of all integration events.
Targeting Success: Only 1 in 10 million cells undergo homologous recombination, and 1 in 100 survivors are correctly targeted.

Question 19

Limitations and Considerations

Answer 19

Inefficiency: Process is inefficient, and early embryonic lethality may hinder later event analysis.
Phenotypic Variation: Mouse strain choice affects observed phenotypes due to interactions with host’s varying genes.