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Modification of YAC Both Arms with Three Genes by Homologous Recombination
Li-Rong Liu1, Bu-Feng Lu*, Zi-Qing Lv, Bo Lu, Xiao-Lei Zhang1, Li-He Guo*
Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Cel-star Institute of Biotechnology, Shanghai 201203, China; 1Guiyang Medical College, Guiyang
Abstract: Yeast artificial chromosome (YAC) was widely used in genomic and chromosome relative researches due to its extraordinary capacity of loading large DNA segments (megabase pairs， Mbp). In this experiment， different vectors bearing YAC homologous sequences and mammalian cell selective genes， such as NEO， EGFP， PURO， were transferred into yeast. By means of homologous recombination， the YACs， which were modified by the genes mentioned above on certain fragments of both arms， were selected and pooled on nutrition-deficient medium， excluded mutation and random integration. Then， the integration of NEO， EGFP and PURO into YAC DNA were identified using PCR and Southern blotting methods. The results indicated that such integration was successfully carried out and the YAC bearing mammalian selective genes was obtained. At the same time， the method， which mammalian selective genes integrated on YAC arms using homologous recombination， was constructed.
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