Knock-out gene mutations Flashcards
lecture 17 (35 cards)
Why are mouse models needed in biomedical research?
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.
What are some common model organisms used in biomedical research?
Mouse (Mus musculus), Clawed frog (Xenopus sp.), Zebrafish (Danio rerio), Fruit fly (Drosophila melanogaster), Nematode worm (Caenorhabditis elegans).
What ethical considerations are involved in animal research?
Animal research is carried out only when no feasible alternative exists. In the UK, animal experiments require three licenses: personal, project, and establishment licenses.
What is the main difference between in vitro models and in vivo mouse models?
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.
What are the main types of genetically modified mice used to study human diseases?
Transgenic mice, Knock-out mice, and Knock-in mice.
How are standard transgenic mice created?
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.
What are the strengths and weaknesses of transgenic mouse models?
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.
What is the gene targeting approach for creating knock-out mice?
Gene targeting involves homologous recombination to precisely disrupt a gene’s function. It requires screening many embryonic stem (ES) cells to find correct recombination.
What is the “GeneTrap” approach?
Gene trapping introduces insertion mutations across the genome in mouse ES cells to disrupt gene function, report gene expression, and identify the insertion site.
How does the Cre-Lox system work for creating conditional knock-outs?
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.
What is CRISPR-Cas9 and how has it revolutionized genetic modification in mice?
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.
How are transgenic mouse models used in disease modeling?
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.
What is an example of using a knock-in mouse model for disease modeling?
The introduction of the V194D mutation in matrilin-3 (MATN3) in knock-in mice to model multiple epiphyseal dysplasia (MED), a genetic skeletal disease.
How are transgenic ‘phenocopies’ used in disease research?
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.
How do mouse knock-out models probe disease mechanisms?
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.
What is a potential outcome of using knock-out models in skeletal disease research?
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).
What is the main characteristic of the mouse as a model organism?
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.
What is a potential downside to using mice as a model organism?
While mice are genetically similar to humans, they are relatively expensive to maintain, especially compared to other model organisms like fruit flies or zebrafish.
What are the steps involved in generating a transgenic mouse model using a simple vector construct?
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.
Why are multiple founder mice generated in transgenic experiments?
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.
What is a key challenge when using transgenic mice for research?
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.
How does the gene targeting approach differ from the standard transgenic approach?
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.
What role does the Cre recombinase play in generating conditional knock-out mice?
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.
What is the advantage of using conditional knock-out mice?
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.