Homologous recombination strategy to directly clone mammalian telomeres. Final progress report, March 15, 1991--March 14, 1994

摘要

The goal of this project was to develop a strategy for the direct molecular cloning of the termini of mammalian chromosomes as Yeast Artificial Chromosomes (YACs). We proposed to use homologous recombination in cultured human cells to introduce a vector (given the acronym HARY) near to chromosomal telomeres. Previous reports described how we constructed the targeting vector to contain a yeast telomere, replication origin, centromere, yeast and mammalian selectable markers and a site for the restriction endonuclease I-SceI. The 18bp, G-C rich target site for this enzyme should only exist in the human genome at the site introduced by the targeting vector because. We then proposed to remove the targeted vector and flanking human sequences from the chromosome by either selection for gene amplification, or by cleavage with I-SceI, followed by amplification and rescue by transfection of yeast.