Encyclopedia of Experiments: Biological Techniques
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CreER-LoxP System-Based Target Gene Inactivation - ExperimentTranscript
Begin with a genetically engineered mouse model comprising a modified genome that harbors LoxP sites - specific sequences recognized by Cre-recombinase enzymes - flanking the gene of interest. The mouse is further transfected with a plasmid vector encoding organ-specific CreER recombinase, leading to the integration of CreER recombinase-expressing element into the genome of liver cells.
The expressed CreER recombinase, an inactive fusion protein involving Cre recombinase and the mutant ligand-binding domain of the estrogen receptor, ER, interacts and binds to heat-shock protein in the cytoplasm. This binding confines CreER recombinase in the cytoplasm.
Prepare a solution of tamoxifen, a hydrophobic drug, using ethanol and suitable oil. Now, inject the tamoxifen solution intraperitoneally into the left lower quadrant of the mouse's abdomen to prevent damage to the abdominal organs.
The injected tamoxifen is metabolized in the liver to 4-hydroxytamoxifen, 4-OHT. Further, 4-OHT, a selective estrogen receptor modulator, binds to the ER-ligand binding domain of CreER recombinase, causing its conformational change. This leads to the release of the CreER recombinase from the heat-shock protein.
The activated CreER recombinases translocate into the nucleus and recognize the LoxP sites in the host genome. The CreER recombinases catalyze site-specific recombination between the LoxP sites, resulting in the excision of the gene of interest, thereby achieving tissue-specific gene knockout.
