In the past, the Tissue Culture Core has been responsible for culturing both keratinocytes and fibroblasts from patients with inherited skin disorders as well as from their relatives and healthy control subjects. The clinical material, which will be obtained from Core E (Clinical and Administrative Core), as part of the clinical evaluation of the patients, will be transported for initiation of the cell cultures to the tissue culture facilities of the Tissue Culture Core of the Department of Dermatology and Cutaneous Biology. This core will also be responsible for maintenance and storage of these cells, as well as cultures being sent to us from other laboratories, so that they are available to the investigators in Projects 1-5. The future goals of this core include increasing the number of cell strains established from patients with different inherited skin disorders. Towards this goal, several of our participating clinical collaborators have pledged their support to continue providing patient material for our studies. We have also solicited several additional investigators who will serve as Consultants to our project. These investigators have expressed their willingness to share their clinical expertise and wealth of clinical material with us (see Core E). This network of international clinician-investigators guarantees the availability of a large number of new patients with distinct phenotypic subtypes to our studies. We have previously established collaborative contacts with the National EB Registry, and our collaboration with the Principal Investigators of the two clinical sites, Dr. Jo-David Fine (University of North Carolina, Chapel Hill), and Dr. Joseph McGuire (Stanford University), will help to ensure continued access to a large number of well-documented patients with EB. In addition, Dr. Uitto, Dr Richard, and other physicians in the dermatology clinic at Thomas Jefferson University expect to see a number of patients with the genetic disorders outlined in Project 1. Dr Richard has also established a foundation of clinical collaborators from whom patients will be referred. The availability of cultured cells from patients, as well as control cells from both unaffected healthy controls and from patients? relatives is essential to the success of projects 1 through 3, and project 5. Projects 1 and 2 require both mutant and normal cells to examine consequences of the genetic alterations at the protein level. Project 2 requires normal cell cultures to provide the mRNA and DNA needed for characterization of newly developed gene probes through Northern and Southern hybridizations. Projects 1 and 2 utilize cells from patients to identify and verify the existence of mutations at the DNA and RNA levels. Project 5 requires mutant and control cells, to test the feasibility of homologous recombination using chimeric RNA/DNA oligonucleotide constructs. This project will also utilize cells to study the expression of gene constructs as part of the testing of delivery systems. Project 1 also requires DNA for genetic linkage analyses. The DNA can be isolated from cultured cells, or from peripheral blood leukocytes, or from epithelial cells obtained by buccal cytobrushing. Due to the relative ease by which blood samples are obtained, peripheral blood will be used as the primary source of DNA, but if blood samples are not available, cell cultures may be required also for Project 1. Although the handling of blood samples, and specifically the extraction of DNA, takes place in Project 1, selected blood samples will be transported to the Tissue Culture Core A for establishment of permanent lymphoblastoid cell lines.
The second aim of the tissue culture core is to immortalize cells from patients with inherited skin disorders, in a manner such that the expression of the BMZ genes is not compromised. This particular function is important to assure availability of mutant keratinocytes from patients with different forms of inherited skin disorders. For protocols currently being utilized for immortalization of keratinocytes, as well as other cell types, see description below.
The third aim of the tissue culture core is to provide DNA extracted from blood and tissues as well as cell cultures for projects 1 and 2 and to establish and maintain a DNA bank from all patient samples submitted for analysis. The DNA extraction and banking functions of the core will enable centralized access to DNA samples and allow retention of samples in the banking freezer for future use by members of the program m project and the dermatological community at large. In addition documents pertaining to patient contact, phenotype and other clinical information will be accessed and filed through the Administrative Core in conjunction with the DNA extraction services of the cell culture core.
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