The pRMCE series of vectors -- development of a repeatedly manipulable transgene system.

This Major Qualifying Project (MQP) involved the construction of a cloning vector that would facilitate repeated manipulation of large DNA fragments in embryonic stem (ES) cells. This system is primarily being developed for use in researching mediators of chromatin regulation, although many other applications could be aided by the technique. Chromatin regulation includes epigenetic phenomenona affecting gene expression such as presence of boundary elements, silencers, DNA methylation, and genomic imprinting. The purpose of the vector is to allow alteration of transgenic constructs containing recombinase target sites, using recombinase-mediated cassette exchange (RMCE), but on a scale of tens of kilobases rather than several kilobases, which is the extent to which the technique has currently been developed. RMCE would be extremely useful in the analysis of complex regions such as the imprinting center (IC) of chromosome 15q11-q13, where an array of regulatory elements is distributed in a region larger than 35 kilobases. The initial template for analysis of the vector will be a 160kb transgene containing the IC. An aim of the system is to analyze whether heterochromatin-associated DNA identified at the IC plays an active role in the initiation of genomic imprinting