Research objectives and content
My research project consists of two independent studies that will be pursued in parallel. The results of the experiments will determine which project will receive emphasis. The objectives of these projects are outlined below:
Analysis of Krox-20 regulation in rhombomere 3 (r3) and rhombomere 5 (r5) DNA centered around the Krox-20 gene has been cloned in three cosmid vectors. I will subclone several fragments into a reporter construct which contains the lacZ gene driven by the B-globin promoter. This promoter lacks specific expression in the developing hindbrain but is very sensitive to enhancer activity. Transgenic mice will be generated and examined for lacZ expression in r3 and r5. Enhancer elements will be localized by deletion mapping and specific binding sites will be identified by homology searches and gel retardation analysis using extracts of mouse or chick embryos. Phenotypic analysis of Krox-20 and Krox-24 double mutants
Double mutant mice defective in both functional Krox-20 and Krox-24 will be generated by crossing mice heterozygous for each gene followed by crossing the double heterozygotes. A detailed phenotypic analysis involving morphologic, histologic well as molecular analyses will be performed to identify phenotypic abnormalities that are unique to the double mutants and may reflect areas of functional redundancy or rescue between these genes. Training content (objective, benefit and expected impact)
We expect that the work on the promoter of Krox-20 will provide a first insight at the factors governing early patterning in the hindbrain. The double mutant studies will complement previous studies on Krox-20 and Krox-24 mutant mice and increase understanding of the role of these transcription factors in development. These studies involve a wide range of techniques that are critical for research in understanding developmental processes. It is my goal to acquire a strong basis of knowledge in the area of vertebrate development. This training in Dr. Charnay's laboratory will form a foundation on which to build my care in this field.