Kouprina N, Kawamoto K, Barrett J C, Larionov V, Koi M
Laboratory of Molecular Genetics, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Genome Res. 1998 Jun;8(6):666-72. doi: 10.1101/gr.8.6.666.
In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in approximately 1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.
与其他动物细胞系不同，鸡前B细胞淋巴瘤系DT40表现出高水平的同源重组，可利用这一特性在特定的靶基因或区域产生位点特异性改变。此外，在DT40细胞系中产生人/鸡单染色体杂种的能力为特异性靶向人类基因开辟了一条途径。在此，我们描述了一种直接分离人类染色体区域的新策略，该策略基于用含有酵母选择标记、着丝粒和ARS元件的载体靶向染色体。通过将总基因组DNA转染到酵母原生质体中，这一过程可拯救靶向区域。对酵母标记进行选择可导致以大小达170 kb的大型环状酵母人工染色体（YAC）形式分离染色体序列，这些YAC包含靶向区域。这些YAC是通过酵母中靶向染色体片段的常见重复序列之间的同源重组产生的。或者，当酵母转化混合物中包含YAC片段化载体时，靶向区域可以作为线性YAC被拯救。由于一旦获得靶插入，染色体区域的整个分离过程大约可在1周内完成，因此该新方法极大地扩展了同源重组能力强的DT40鸡细胞系统的实用性。
