Towards establishment of higher-order of nuclear architecture analysis method by using a human artificial vector

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In order to establish a novel method to analyze a higher-order nuclear architecture, we previously developed a human artificial (HAC) vector which behaves as an independent mini-chromosome and allows the analysis of visualization of the loaded genome on it through LacO tandem on HAC. We focused on the HoxB gene cluster because the genes dramatically alter its nuclear territory during development with on-off of the transcription and this phenomena is known to be reproducible in neuronal differentiation from ES cells. We obtained BAC-based genome for the locus and managed to tag a loxP site to allow the loading of it onto HAC, by BAC-recomibineering. We observed that the HoxB gene cluster was partially deleted/mutated by an unwanted manner specifically in a bacterial strain used for BAC-recombineering most likely due to its highly repetitive sequence and could not carry out a LoxP-tagging in a wanted manner. Thus, we planned to directly introduce human Chr17 carrying human HoxB gene cluster into mouse ES cells by chromosome transfer method, following homologous recombination-mediated tagging of sequence to allow visualizing. We established a method to transfer human Chr17 into mouse ES cells method and obtained mouse ES cell clones carrying human Chr17 and observed neuronal differentiation of such cell clones by retinoic acid. However, we observed the same phenomena as above-unwanted deletion specifically occurred in mouse RS cells. Therefore, we presented that highly repetitive sequence is extremely difficult to deal in recombination-proficient bacterial and animal mills and propose that the alternative way to analyze such genes is to tag a sequence to allow visualization next to the endogenous gene of interest in ES cells