23 Questions
What is a key advantage of using mice as a preclinical model?
Ease of modifying the genome of their embryonic stem cells
What is a characteristic of inducible and tissue-specific mouse models?
They allow for targeted gene expression in specific tissues
What is a key advantage of using mice as a preclinical model in terms of their genome?
They are easy to modify at the embryonic stem cell level
What is the purpose of transgenesis in mice?
Studying protein fate and biological function
Which approach is used to introduce a transgene or create a knockout in mice by inserting a neo-cassette in a critical part of a gene?
Homologous recombination
What is the first step to create a knockout mouse model?
Culturing embryonic stem cells derived from mouse trophoblast
What is the purpose of knockout mice?
Studying the impact of loss of function on phenotype
Which gene is added beyond the sequence of the target gene in knockout mice?
Thymidine kinase gene from HSV virus
What is the mechanism of action of the TKHSV gene?
Production of a toxic nucleotide inhibiting DNA synthesis
What is the purpose of introducing a stop codon in exon 8 for PINK1 gene knockout?
To disrupt the translation of the PINK1 gene
What is the role of neomycin antibiotics in creating knockout mouse models?
To eliminate non-recombinant ES cells lacking the Neomycin resistance cassette
What is the function of single guide RNA in the CRISPR-Cas9 system?
Directing the CRISPR-Cas9 system to the target spot in the genome
What is the purpose of negative selection with ganciclovir in creating knockout mouse models?
To kill non-homologous recombinants carrying the TKHSV gene
What triggers the movement of Cre into the nucleus to excise a gene in the Cre-lox recombination system?
Tamoxifen
What is used to visualize gene expression and then removed in gene knockout research?
LacZ tag and Neo cassette
What is used in the knockout first strategy for gene targeting?
Gene trap targeting cassette and Flp sites
What enables efficient and flexible gene targeting in mouse models?
Knockout first strategy
What is used to confirm the floxed allele and Cre expression in gene knockout research?
Genotyping mice
What is the mechanism by which CRISPR-Cas9 allows for precise gene editing at the site of DNA cleavage?
Homologous Recombination (HR)
What does the Cre/lox system enable in terms of knockout models?
Tissue-specific and/or inducible knockout models
How can mice expressing the Cre recombinase gene be generated?
Using the transgenic embryonic stem cell injection method for tissue-specific knockout models
What is the function of the CreERT2 model in inducible knockout models?
Provides control over the tissue targeted and the timing of knockout
What is the role of the LoxP flanked gene in mice crossbreeding with the Cre mouse?
Approximately 50% of the offspring being heterozygous for the loxP allele and hemizygous for the Cre transgene
Study Notes
CRISPR-Cas9 and Tissue-Specific Genome Editing
- CRISPR-Cas9 induces DNA repair via Non-Homologous End-Joint repair (NHEJ) leading to insertion or deletion in the double strand break (DSB) and resulting in a knockout.
- CRISPR-Cas9 can also utilize Homologous Recombination (HR) for precise gene editing at the site of DNA cleavage.
- HR allows for the introduction of specific point mutations to mimic genetic diseases or modify aspects of the encoded protein.
- It is possible to replace the endogenous gene by an entirely different genetic sequence using CRISPR-Cas9.
- CRISPR/Cas9 can cut dsDNA in a specific way without disrupting the gene sequence, allowing for knock-in of specific sequences.
- The Cre/lox system allows for tissue-specific and/or inducible knockout models, avoiding embryonic lethality in crucial gene knockout cases.
- Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage, performing site-specific recombination between LoxP sites.
- Mice carrying a LoxP flanked gene on two alleles can be generated using the transgenic embryonic stem cell injection method.
- Mice expressing the Cre recombinase gene can be generated using the transgenic embryonic stem cell injection method for tissue-specific knockout models.
- Crossbreeding the Cre mouse with the floxed mouse results in approximately 50% of the offspring being heterozygous for the loxP allele and hemizygous for the Cre transgene.
- Inducible knockout models, such as the CreERT2 model, utilize a mutated estrogen receptor (ER) fused to Cre as a transgene, activated by 4-hydroxytamoxifen (4-OHT).
- The CreERT2 model provides control over the tissue targeted and the timing of knockout, with activation and translocation of Cre upon binding of the active tamoxifen metabolite.
Test your knowledge of CRISPR-Cas9 and tissue-specific genome editing with this quiz. Explore concepts such as Non-Homologous End-Joint repair, Homologous Recombination, Cre/lox system, transgenic embryonic stem cell injection, and inducible knockout models.
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