Tucker R M, Burke D T
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109, USA.
Gene. 1997 Oct 15;199(1-2):25-30. doi: 10.1016/s0378-1119(97)00306-5.
The introduction of cloned DNA into mammalian cells allows functional testing of genes contained on the fragments. In many cases, the exogenous DNA introduced into mammalian cells requires selectable genes that mark the presence of input DNA. Two new vectors, carrying mammalian selectable markers encoding for either neomycin-resistance (neo) or histidinol-resistance (hol), have been constructed for targeted integration to specific single-copy sites within yeast artificial chromosome (YAC) insert DNA. The integration cassettes comprise a single selectable yeast gene adjacent to a mammalian selectable gene, either LEU2 with neo or HIS3 with hol. Modification of the YAC occurs in yeast by transfection with linear DNA containing YAC-specific, unique, recombinogenic ends, thereby ensuring co-integration of the markers. Analysis of modified YACs confirms that both vectors correctly integrate into the targeted unique sites. The precise localization of selectable marker genes in the cloned DNA ensures the integrity of the genomic fragments during functional testing. Placement of mammalian selectable markers within the YAC insert DNA should allow for YAC-based gene targeting experiments in a variety of mammalian cell lines.
将克隆的DNA导入哺乳动物细胞可对片段中所含基因进行功能测试。在许多情况下，导入哺乳动物细胞的外源DNA需要可选择基因来标记输入DNA的存在。已构建了两种新载体，它们携带编码新霉素抗性（neo）或组氨醇抗性（hol）的哺乳动物选择标记，用于靶向整合到酵母人工染色体（YAC）插入DNA内的特定单拷贝位点。整合盒包含一个与哺乳动物选择基因相邻的单一可选择酵母基因，即与neo相邻的LEU2或与hol相邻的HIS3。通过用含有YAC特异性、独特、重组性末端的线性DNA转染，在酵母中对YAC进行修饰，从而确保标记的共整合。对修饰后的YAC的分析证实，两种载体都正确整合到了靶向的特定位点。可选择标记基因在克隆DNA中的精确定位确保了功能测试期间基因组片段的完整性。在YAC插入DNA中放置哺乳动物选择标记应允许在多种哺乳动物细胞系中进行基于YAC的基因靶向实验。
