During fiscal year 2010 we accomplished the following: 1) We obtained G418-resistant clones of ES cells that had been transfected with 4 different targeting constructs. Three of these introduce LacO or TetO sites at different locations in the IgH locus and the fourth one substitutes Eμsequences with both LacO and TetO sites. We obtained 50-60 clones with each vector for a total of 200-250 clones. Genomic DNA from these clones is being analyzed by Southern blotting to identify clones that have undergone homologous recombination. 2) We recombineered 2 bacterial artificial chromosomes (BACs) to introduce LacO sites. One of these BACs contains the 4 tandem copies of L1 transposons and one of them does not;both BACs are derived from different parts of the VH locus. The recombineered BACs also contain a Neo gene that is flanked by lox P sites. Once stable clones have been obtained in mouse embryo fibroblasts, transient cre expression will be used to eliminate Neo to minimize artefacts cause by its presence. Our objective is to study by FISH whether L1 repeats affect radial nuclear position of these genes. 3) To continue our analyses of telomere position effects (TPE), we expanded and froze two lines of human diploid fibroblasts that had been grown for different time periods. As expected low PD (population doubling) cells have longer telomeres and high PD lines have shorter telomeres. We also obtained a clone that continuously expresses hTert;telomere length in this clone does not shorten with extended growth. Chromatin prepared from low PD and high PD cells will be used for immunoprecipitation with antibodies against heterochromatic marks associated with telomeric silencing. Our objective is to unequivocally establish whether the extent of telomeric heterochromatin changes as a function of telomere length.
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