| Popis: |
Understanding DNA repair mechanisms for ionizing radiation (lR)-induced DNA damage and having prior knowledge of a patient's IR-specific repair capacity will help determine which patients will be most responsive to radiation therapy and design more effective treatment regimes. The objective of this work has been to define the contributions of the mammalian protein Apel, and other candidate nucleases, to the repair of IR- induced genetic damage. We are currently constructing cell lines that lack Apel protein and will determine the sensitivity of these mutant cells to various DNA-damaging agents, particularly IR. We have constructed a hamster CEO cell line that has conditional expression of the human Apel protein under the control of a tetracycline-responsive promoter (Tet-off system of Clonetech). These cells are being used in gene targeting DNA transfection experiments to obtain a knockout mutation in Apel. Extensive analysis has been done to design highly sensitive PCR (polymerase chain reaction) screening procedures to identify and recover cells have Apel recombination events. So far, we have seen numerous gene targeting events that involve recombination of the right arm, but not the left arm, of the targeting vector. Ongoing studies are focused aggressively on getting the knockout mutant and performing extensive characterization of its radiation responses. |
