CRISPR Protocol

Proliferation is an important part of cancer development and progression. This is manifested by the expression and / or altered activity of proteins related to the cell cycle. The constitutive activation of many signal transduction pathways also stimulates cell growth. Once tumors appear, growth and metastasis can be supported by the overproduction of appropriate hormones (in hormone-dependent cancers), by promoting angiogenesis, by the transition from epithelium to mesenchymal, by triggering autophagy and by take the signals of the surrounding stromal cells. Cancer therapy, which involves cytotoxic drugs, destroys cells that have a high basal level of proliferation and regeneration. While this type of therapy targets tumor cells, it affects non-tumor cells that proliferate rapidly. The objective of this study is to program through CRISPR-Cas9 a sequence to genetically inhibit the proliferation pathway of the rapamycin Diana protein in mammalian cells (mTOR) to alter and block the proliferation of tumor cells by coupling it to the target cells by electroporation medium.

The mTOR protein is involved in the control of the initiation of RNA transcription, organization of the actin cell cytoskeleton, membrane traffic, ribosome formation and growth regulation, cell proliferation.

The two proteins that interact with mTOR, raptor and rictor, define the different arms of the mTOR path. The raptor-mTOR pathway regulates cell growth (accumulation of cell mass) through S6K1 and 4E-BP1. It responds to nutrients and growth factors, partly due to regulators such as TSC1 / TSC2 and rheb. The rictor-mTOR complex regulates Akt / PKB, PKCα, Rho / rac, to control cell survival, proliferation, metabolism and cytoskeleton. The binding of growth factors to receptors on the active PI3K cell surface, to generate PIP3, which recruits the PKD1 and Akt / PKB kinase to the membrane. Akt / PKB is activated by phosphorylation in two different places. The rictor-mTOR complex phosphorylates Akt / PKB in serine 473, in a hydrophobic motif, which facilitates phosphorylation by PKD1 in the Akt / PKB activation loop in threonine 308.

Neoplastic cells require a large cocktail of cytokines for cell cycle control, proliferation and metastasis. Inhibitors directed against the metabolic pathways generated by these cytokines are generating promising results in the treatment of solid tumors and leukemia. As mentioned earlier, the mTOR protein seems to be an essential protein that is in the PI3K signaling pathway. On the other hand, there is solid experimental evidence that reinforces the role of mTOR as a key regulator of the cell cycle, directing expansion, growth and proliferation.

Goals:

Generate a vaccine using CRISPR Cas9 to couple an interference sequence to P42345 that represents the mTOR genetic code.

Methodology:

An interference sequence will be designed for the initiators and part of the mTOR coding sequence in order to couple it to the CRISPR-Cas9 system and generate a selective genetic inhibitor of cell proliferation.

To evaluate the effectiveness of the designed system,

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