The overall function of the Cell and Molecular Biology (CMB) Core will be to provide the program withcarefully controlled and characterized human cell cultures that are needed in each project. Servicesprovided by the core wjll include the isolation, culture, storage and distribution of primary melanocytes,keratinocytes and fibroblasts. In addition, the CMB Core will also culture and distribute all of the melanomacell lines that will also be characterized for DNA mutations in B-RAF, N-RAS and PTEN. The CMB Core willensure that all cells used in the program are free of contamination with mycoplasma or other contaminantsby providing regular testing and decontamination when necessary.The CMB core will generate virus fortransduction of primary melanocytes with melanomagenic genes or shRNA's to knockdown DNAdamageresponse genes. Transduced cells will be characterized for expression and function of these genes andknockdown vectors using informative assays such as western blot analysis. The CMB Core will generate invitro human skin reconstructs (also referred to as orgariotypic skin cultures or skin equivalents) in whichartificial human skin is created in vitro in a three-dimensional system with a mixture of primary melanocytes,keratinocytes and fibroblasts. These skin reconstructs will be supplied to Project 1 for in vitro analysis ofDNA damage response and Project 3 for engraftment on the backs of SCID mice for in vivo analysis of DNAdamage response. The PI of the CMB Core (Dr. Shields) recently completed an NCI-sponsored two-weektraining program at the Wistar Institute with Dr. Meenhard Herlyn (an internationally recognized leader inmelanoma research and Program externaladvisor) to gain hands-on training in preparation of human skinreconstructs. The Cell and Molecular Biology core serves the Program Project by providing importantbiological reagents to program investigators.
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