Current technology for the isolation of mammalian cell mutants which are sensitive to environmental agents are extremely laborious and time-consuming, precluding the ability to screen large numbers of clones. In the specific case of radiation, there are less than 20 sensitive mammalian cell clones reported in only a few complementation groups. We are developing a fundamentally new procedure for rapidly isolating mammalian cell mutants which are sensitive to radiation. This technique combines the culture of large numbers of clonally-derived, three-dimensional mammalian cell aggregates (spheroids) with methods we have developed for rapidly analyzing and sorting intact spheroids using a flow cytometer. This procedure will have several advantages over current mutant isolation procedures 1) rapid isolation of large numbers of individual clones for sensitivity testing; 2) automated sensitivity analysis; 3) rapid separation of sensitive clones; and 4) recovery of surviving cells directly from the same colonies which are tested for sensitivity. We have demonstrated all of the steps critical to this procedure, and radiation sensitive and resistant cell lines for calibration and verification of the entire isolation method are available. We will construct and test a flow cytometer for sorting spheroids on the basis of size and improve our methods for obtaining large numbers of clonal spheroids. The measurement and culture parameters will be optimized for the separation of rare sensitive clones in the second year. The third year will involve the isolation and characterization several new radiation-sensitive mutants from standard cell lines. This method could be used to screen 107 clones for sensitivity in one day, representing an improvement of 1000-fold over current technology. Importantly, the ability to isolate new radiation-sensitive mammalian cell mutants will assist efforts to characterize the molecular mechanisms behind the repair of ra diation-induced DNA damage. While radiation sensitivity willbe used as the endpoint for testing and initial application, this new technique will be applicable to essentially any DNA damaging agent.
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