Lecture 4 – Conditional knockouts Flashcards
Early Embryonic Deaths in Knockouts
Challenge: Mice dying in early embryogenesis due to gene knockout.
Implication: Unable to study gene knockout effects in specific regions of adult mice.
Solution 1: Make chimeric mice by aggregating mutant and wild-type embryos or injecting mutant ES cells into a wild-type blastocyst.
Solution 2: Use Cre/loxP technology for tissue-specific knockout.
Cre/loxP Technology
Cre Recombinase: Site-specific enzyme recognizing 34 bp DNA sequence loxP.
Function: Snips out target DNA when loxP sites are introduced.
Alternative: Flp recombinase recognizes FRT sites, performs the same function in mice.
Mouse Strains: One with the target gene flanked by loxP sites (floxed), and another expressing Cre recombinase from a tissue-specific promoter.
Breeding Outcome: Offspring with knockout only in tissues where Cre is expressed.
Floxed Mice Creation
Process: Homologous recombination in ES cells, replacing the endogenous gene with a floxed version.
Selection: Floxed version has neomycin resistance gene and tyrosine kinase for selection.
Chimeric Offspring: Successfully edited ES cells injected into blastocysts, implanted in female mice to create chimeric offspring.
Heterozygous Floxed Mice: Chimeric mice bred to produce mice with a floxed endogenous gene copy without the neomycin resistance gene.
Cre Expression in Tissues of Interest
Methods: Cre expressed in the tissue of interest (rare), or in areas overlapping with the tissue of interest.
Alternative: Create a knock-in where the Cre gene is inserted into a gene expressed in the tissue of interest.
Conditional Knockout: Mice with floxed gene bred with mice producing Cre for targeted knockout.
Control of Cre Expression
Drug Control: Tetracycline and tamoxifen used to turn Cre on or off by putting them into the drinking water of mice.
Tet-Off System: TetO (tet operon) is a promoter of Cre, requiring TetA protein for functionality.
Tet-On System
Mutation: One amino acid difference in Tet activator protein changes its activity.
Tet-On Activation: Mutant TetA stays in the cytoplasm until tetracycline is added.
Cre Production: Tetracycline addition activates TetA, leading to Cre production.
Cre Function: Activated Cre acts on floxed genes.
Tet-Off System
Tetracycline Control: TetA protein, under TetA constitutive promoter, binds to tetracycline, staying in the cytoplasm.
Activation: Without tetracycline, TetA moves to the nucleus, activating TetO for Cre production.
Cre Function: Activated Cre acts on floxed genes.
Continual Tetracycline: Prevents activation of the Cre promoter, keeping the gene unfloxed.
Tamoxifen-Inducible Cre Expression
Tamoxifen Use: Tamoxifen induces Cre expression.
Tamoxifen Function: Antiestrogen hormone, binds to the mutant form of the estrogen receptor (ERTM).
In the Absence of Estrogen: ERTM stays in the cytoplasm, no gene expression changes.
In the Presence of Estrogen: ERTM moves to the nucleus, altering gene expression.
Tamoxifen Exploitation: Cre is bound to ERTM, and tamoxifen induces Cre-mediated recombination by releasing Cre-ERTM from Hsp90 in the nucleus.
