The Cellular and Molecular Biology Core (CMBC) will provide services to the CCEMH members using histology, immunohistochemistry (IHC) and mRNA in situ hybridization in their studies of transgenic models, as well as in the lines are being produced in the analysis of hematopoietic genes and diseases, as well as in the studies of gene therapy protocols, the ability to characterize the expression in situ, both for mRNA and protein, is becoming and more a necessity to correctly ascertain the function in these types of experimental paradigms. Furthermore, the number of these types of analyses is projected to increase rapidly, outstripping the ability of independent investigators to perform these analyses in a cost-effective or timely manner. A cohesive, efficient, and coordinated CMBC, will greatly decrease the cost and increase the reliability for these analyses to be performed for members of the CCEMH. Furthermore, as the efficiency of gene transfer improves in the future, a critical component in the evaluation of gene transfer protocols will be quantitation of transgene expression and assessment of biochemical endpoints. The Cell and Molecular Biology Core will not only assist a large number of investigators of the CCEMH working with transgenic and knock-out mice, but will also provide essential services to pre-clinical and clinical gene transfer research. The core will provide: 1) standardized processing, embedding and staining of tissues for histological analysis, 2) sectioning, embedding (paraffin or frozen) and IHC processing. Also, antibody characterization (Western blotting) and titer analysis will be performed for specificity and quality control of the antibodies used, 3) mRNA in situ analysis will be performed on frozen sections. Sense and anti-sense probes will be produced, characterized and quality controlled for each gene probe generated. Either 35S or non-radioactive probes will be used as per the investigators request, and 4) apoptosis assays on cell cytospins or tissues will be performed using the TUNEL assay on frozen or paraffin sections. Double labeling experiments can be performed using an antibody for specific gene product along with the TUNEL assay if required. Altogether, the core will provide accurate, efficient and cost-effective analysis of gene expression in vivo, with substantial savings compared to having each individuals investigator performing these analyses independently.
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