Question

Your colleague in the lab next door is studying the disease cerebral cavernous malformations (CCM) and discovers that the human homologue of the worm exc12 gene (human gene is HEXC12) is mutated in a small fraction of familial CCM patients. You decide to collaborate to test if this gene has a CCM phenotype in mouse, cultured cells, and zebrafish models.

1. Explain how you would generate a mouse model.

2. Based on its sequence you hypothesize that HEXC12 encodes as a small GTPase exchange protein (GEF) that might regulate the activity of RhoA or CDC42. Explain how you would test this hypothesis using cultured cells.

3. How would you determine if HEXC12 regulates the activity of the Rho kinase (ROCK) using cultured mammalian cells?

4. How would you determine if HEXC12 is required in the endothelium or surrounding smooth muscle for proper vascular development in the mouse?

5. To gain more insight into HEXC12 you decide to use zebrafish as a model system. Explain how you would determine if it has a CCM phenotype in fish.

Answer 1

My colleague is studying the disease, cerebral cavernous malformations which is a result of defective or abnormal blood vessels in the brain. The capillaries are extremely thin and when blood flows in through them, they often leak resembling caverns. This results in an altered flow of blood which gives rise to symptoms such as seizures, headache, paralysis and even cerebral hemorrhage in severe cases. This disease is a result of mutations in several genes, like the CCM1, CCM2 and the CCM3 gene. Apart from these in the study, my colleague scientist observed that there is an alteration in the HEX 12 gene as well which is a human homolog of the ex 12 genes found in worms.

1. The first step would be the generation of the mouse model. The mouse model is a setup which is done in laboratories to study human disease. Here the disease which is being studied is CCM. The mouse model involves a mouse that is genetically modified by inserting a vector that has the required gene of interest. This gene of interest is then transferred in the embryonic stem cells and then the mouse is grown and carried out for scientific interests.

2. HEX 12 gene which is the human homolog of the ex 12 genes found in the worm is a GTPase exchange protein and it targets the Rho A. Rho A is activated by them by the process of phosphorylation. Rho A is a group of G proteins and their function lies in the cytoskeletal organization. In order to observe this hypothesis in the cultured media, we take the help of immunocytochemistry. In the process of immunocytochemistry, detection is observed when phosphorylation of the GTPase occurs.

3. Rho-associated protein kinase( ROCK) is a group of kinases that belongs to the group of serine-threonine. The function of this group of the kinase is again to regulate the cytoskeletal functions. The hypothesis here is that HEX 12 regulates the function of ROCK. In order to prove this hypothesis, we use the ROCK activity assay kit made by Millipore. This assay kit is handy and easy to use as it has pre-coated wells which detect the phosphorylation process.

4. The next hypothesis is that the HEX 12 gene helps in the development of endothelium and surrounding smooth muscle of the mouse. In order to have a detailed understanding of the vasculature development of the mouse, we have to study the mouse in its embryonic stages when the initial stages of vasculature begin. To observe that we need to have a 3-dimensional view that is made possible by tomography methods is used to observed the immunostained vasculature of the mouse. In this case, Optical Projection Tomography is the method used.

5. To gain more insight, now the colleague scientist uses HEX 12 gene into zebrafish as a model organism. The uniqueness of zebrafish is that it is close to humans in the genetic constitution. Hence, it is a great model for studying human disease conditions such as CCM. To prove it hypoxia tolerance of the fishes is observed as a disease phenotype.