Knock-out gene mutations

Question 1

Why are mouse models needed in biomedical research?

Answer 1

Mice are genetically similar to humans (~95%) and are used to study human diseases, developmental biology, and gene function. They are ideal for genetic manipulation and disease modeling.

Question 2

What are some common model organisms used in biomedical research?

Answer 2

Mouse (Mus musculus), Clawed frog (Xenopus sp.), Zebrafish (Danio rerio), Fruit fly (Drosophila melanogaster), Nematode worm (Caenorhabditis elegans).

Question 3

What ethical considerations are involved in animal research?

Answer 3

Animal research is carried out only when no feasible alternative exists. In the UK, animal experiments require three licenses: personal, project, and establishment licenses.

Question 4

What is the main difference between in vitro models and in vivo mouse models?

Answer 4

In vitro models study individual cells or organs, but they can’t replicate the complex interactions between cell types that occur in a whole organism, which in vivo models, like mice, can.

Question 5

What are the main types of genetically modified mice used to study human diseases?

Answer 5

Transgenic mice, Knock-out mice, and Knock-in mice.

Question 6

How are standard transgenic mice created?

Answer 6

DNA of interest is microinjected into the pro-nucleus of fertilized mouse oocytes, and these oocytes are then implanted into a pseudo-pregnant recipient mouse.

Question 7

What are the strengths and weaknesses of transgenic mouse models?

Answer 7

Strengths: Relatively easy and cheap to create, can express tagged human genes.
Weaknesses: Cannot control where the transgene integrates, may have variable expression, and the wild-type gene may still be expressed, interfering with the phenotype.

Question 8

What is the gene targeting approach for creating knock-out mice?

Answer 8

Gene targeting involves homologous recombination to precisely disrupt a gene’s function. It requires screening many embryonic stem (ES) cells to find correct recombination.

Question 9

What is the “GeneTrap” approach?

Answer 9

Gene trapping introduces insertion mutations across the genome in mouse ES cells to disrupt gene function, report gene expression, and identify the insertion site.

Question 10

How does the Cre-Lox system work for creating conditional knock-outs?

Answer 10

A floxed allele is created in one mouse and then crossed with a transgenic mouse expressing Cre recombinase under a tissue-specific promoter. This leads to tissue-specific gene deletions.

Question 11

What is CRISPR-Cas9 and how has it revolutionized genetic modification in mice?

Answer 11

CRISPR-Cas9 is a bacterial immune system repurposed to edit genes. It allows for precise DNA editing in mice in a much faster and cheaper way compared to traditional methods.

Question 12

How are transgenic mouse models used in disease modeling?

Answer 12

Transgenic models, such as those created for genetic skeletal diseases, are used to model human diseases by introducing specific mutations into the mouse genome to study the disease mechanisms.

Question 13

What is an example of using a knock-in mouse model for disease modeling?

Answer 13

The introduction of the V194D mutation in matrilin-3 (MATN3) in knock-in mice to model multiple epiphyseal dysplasia (MED), a genetic skeletal disease.

Question 14

How are transgenic ‘phenocopies’ used in disease research?

Answer 14

Transgenic ‘phenocopies’ involve expressing a mutant gene (e.g., thyroglobulin under cartilage-specific promoters) to study disease mechanisms and protein aggregation in tissues like cartilage.

Question 15

How do mouse knock-out models probe disease mechanisms?

Answer 15

Knock-out models, such as those involving Xbp1 and Matn3 genes, are used to study the effects of gene loss and test the role of specific pathways like the UPR (unfolded protein response) in diseases like chondrodysplasias.

Question 16

What is a potential outcome of using knock-out models in skeletal disease research?

Answer 16

Knock-out models can be used to assess the impact of protein misfolding, such as ER stress, in diseases like achondroplasia and multiple epiphyseal dysplasia (MED).

Question 17

What is the main characteristic of the mouse as a model organism?

Answer 17

Mice share approximately 95% of their genes with humans, have a short life cycle, and are genetically well-characterized, making them ideal for rapid breeding and genetic studies.

Question 18

What is a potential downside to using mice as a model organism?

Answer 18

While mice are genetically similar to humans, they are relatively expensive to maintain, especially compared to other model organisms like fruit flies or zebrafish.

Question 19

What are the steps involved in generating a transgenic mouse model using a simple vector construct?

Answer 19

The construct typically includes the gene of interest, a relevant promoter, a 3’ protein tag for detection, and a poly A tail. These components are introduced into the mouse genome via microinjection into fertilized oocytes.

Question 20

Why are multiple founder mice generated in transgenic experiments?

Answer 20

Generating multiple founder mice helps to characterize various mouse lines, as different founders may express the transgene at varying levels, allowing researchers to identify the most suitable line for study.

Question 21

What is a key challenge when using transgenic mice for research?

Answer 21

A key challenge is the inability to control the site of transgene integration into the genome, which can result in varying gene expression levels and off-target effects.

Question 22

How does the gene targeting approach differ from the standard transgenic approach?

Answer 22

The gene targeting approach uses homologous recombination to precisely insert or disrupt genes, whereas the standard transgenic approach introduces the gene randomly into the genome, often leading to variable expression.

Question 23

What role does the Cre recombinase play in generating conditional knock-out mice?

Answer 23

Cre recombinase catalyzes site-specific recombination between two loxP sites, allowing for the deletion of a “floxed” allele in a tissue-specific manner when combined with a tissue-specific Cre transgenic mouse.

Question 24

What is the advantage of using conditional knock-out mice?

Answer 24

Conditional knock-out mice allow researchers to delete specific genes in particular tissues or at specific time points, enabling more accurate modeling of diseases that involve complex tissue-specific gene expression.

Question 25

How can the Cre-Lox system be used in inducible knock-out mice?

Answer 25

Inducible knock-out models involve using a tamoxifen-responsive Cre recombinase, allowing researchers to trigger the deletion of a floxed gene at a controlled time after treatment.

Question 26

What is the significance of the International Mouse Phenotyping Consortium (IMPC)?

Answer 26

The IMPC provides a valuable resource for researchers by offering gene trap lines for virtually all genes, which enables large-scale, high-throughput gene knockout studies in mice.

Question 27

What is the difference between dominant negative mutations and loss of function mutations in mouse models?

Answer 27

Dominant negative mutations result in a gene product that interferes with the normal function of the wild-type allele, while loss-of-function mutations result in the complete absence or inactivity of the gene product.

Question 28

How has CRISPR-Cas9 changed the creation of knock-in mouse models?

Answer 28

CRISPR-Cas9 has made it much faster and cheaper (from 18 months and £20K to about 2 months and a few hundred pounds) to create knock-in mice with specific human mutations for disease modeling.

Question 29

What is the primary application of transgenic mouse models in disease research?

Answer 29

Transgenic mouse models are commonly used to investigate the mechanisms of human diseases, such as genetic skeletal diseases, by introducing specific mutations that mimic human disease phenotypes.

Question 30

What disease was modeled using a knock-in mouse with a mutation in the MATN3 gene?

Answer 30

Multiple epiphyseal dysplasia (MED), a genetic skeletal disorder, was modeled by introducing the V194D mutation in MATN3.

Question 31

What phenotype was observed in mice with the V194D mutation in MATN3?

Answer 31

The mice with the V194D mutation exhibited a short limb dwarfism phenotype, reduced cell proliferation, and increased apoptosis in cartilage.

Question 32

How are transgenic mouse models used to understand disease mechanisms?

Answer 32

Transgenic mouse models express mutant genes under specific tissue promoters to mimic human diseases and study the resulting pathologies, such as toxic protein aggregation or ER stress in skeletal diseases.

Question 33

How does ER stress play a role in diseases modeled in transgenic mice?

Answer 33

ER stress, caused by misfolded proteins in the endoplasmic reticulum, can lead to apoptosis and contribute to disease phenotypes in genetic skeletal diseases like achondroplasia and multiple epiphyseal dysplasia.

Question 34

What did the study involving Xbp1 deletion in Matn3 V194D mice demonstrate?

Answer 34

The study demonstrated that deleting Xbp1, a key regulator of the unfolded protein response (UPR), in Matn3 V194D mice could restore bone growth, highlighting the role of the UPR in managing protein misfolding in diseases.

Question 35

What is the role of Xbp1 in the context of chondrodysplasias?

Answer 35

Xbp1 is part of the UPR pathway, and its deletion in Matn3 V194D mice restored bone growth, suggesting that Xbp1 and the UPR may act as a protective response to protein misfolding in cartilage cells.