This study investigates the role of the CCM2 gene in blood vessel stability using genetically modified mouse pups. The disruption of this gene leads to the formation of fragile vascular structures known as cerebral cavernous malformations (CCMs).
Begin with a genetically modified mouse pup carrying the CCM2 gene, which is essential for blood vessel stability.
This gene is flanked by two loxP sites. The pup's endothelial cells also express Cre recombinase enzyme fused to an estrogen receptor or ER.
Intragastrically inject 4-hydroxytamoxifen or 4-HT into the pup.
The 4-HT diffuses through endothelial cells and binds to the estrogen receptor portion of the Cre-ER fusion protein.
This binding triggers a conformational change that activates Cre recombinase, which enters the nucleus of endothelial cells and binds to the loxP sites.
This facilitates the recombination event between the loxP sites, forming a loop and excises the CCM2 gene segment.
The loss of CCM2 disrupts the integrity of endothelial cells, compromising the stability of blood vessel walls.
This leads to the development of swollen abnormal vascular structures known as cerebral cavernous malformations or CCMs, which are fragile and prone to leakage.
Begin by dissolving 4-HT in 100% ethanol to a concentration of 10 milligrams per milliliter. Prepare 30 microliter aliquots, and then store at minus 80 Celsius. On the day of use, dilute the aliquoted 4-HT in corn oil to 0.5 milligrams per milliliter. Then, use an insulin syringe to intragastrically inject 50 microliters of 4-HT into P1 neonatal pups to induce experimental CCM lesions.
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