Core: Molecular biology

点击翻译按钮获取中文摘要

Adapted from the Applicant?s Abstract) All of the individual projects proposed in this overall program project incorporate the use of molecular biology techniques to answer specific scientific questions addressed in various physiological models. A molecular biology core facility will provide the resources and expertise to carry out these techniques. A molecular biology laboratory and tissue culture facility were established in the Division of Physiology in February 1994. The molecular biology laboratory is well equipped. It is a separate laboratory space containing standard equipment such as a Sorvall centrifuge, spectrophotometer, fluorometer, two hybridization ovens, an Eppendorf PCR machine, a genomyxLR DNA sequencer, a bacterial shaker and incubator as well as adequate bench space, water baths etc. In addition a gel scanner and video camera are set up for image documentation and quantitative analysis of experiments. The tissue culture facility contains two biosafety cabinets, and two CO2 incubators one of which has the capability of controlling O2 levels, and a phase contrast microscope with video camera attachment connected to the imaging system. The tissue culture facility is a biosafety 2 level facility with modification for adenovirus propagation. Therefore, it is appropriate for culture of human cell lines and production of recombinant adenoviruses and retroviruses. To date many techniques have been carried out by members of the Division. Several cell lines are routinely cultured including primary pulmonary fetal fibroblasts and L8 skeletal muscle cells as well as viral packaging cell lines. Cell proliferation assays are accomplished through measurement of total DNA by fluorometry and DNA synthesis by 3h thymidine incorporation. Gene expression at the RNA level is currently analyzed by Northern analysis and quantitative RT-PCR methods are used for small samples sizes such as human biopsies, carotid body samples and single muscle fibers. The laboratory is also set up to measure transcriptional activity by nuclear run-on analysis and transient transfection assay. At the protein level Western analyses are routinely performed for a number of proteins and tissues and procoliagen synthesis is currently measured using the bacterial collagenase digestion assay although an BPLC is available in Dr. Powell?s laboratory for the separation of hydroxyproline and proline in radioactively pulsed lung tissue. Ongoing, in conjunction with the morphology/morphometry core facility, in situ hybridization analyses as well as immunohistochemistry using both color and fluorescent detection systems are performed. Bacterial cells are routinely cultured for subcloning, isolation and purification of CDNA plasmids used as probes in northern analyses, in situ hybridization techniques as well as conditional knock out experiments which are under way. Finally, the technique of differential display and DNA sequence analysis to identify new mRNA species alternatively expressed in tissue and cell experiments is in progress. The laboratory has trained several members of the division in these techniques including technical staff, graduate students, postdoctoral fellows and visiting scientists. The core facility is directed by Dr. Ellen Breen who has extensive experience in molecular biology techniques. Listed below is a table of the projects which will make use of the Molecular Biology Core Facility and the main techniques that will be employed.