Cai L, Schalkwyk L C, Schoeberlein-Stehli A, Zee R Y, Smith A, Haaf T, Georges M, Lehrach H, Lindpaintner K
Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Genomics. 1997 Feb 1;39(3):385-92. doi: 10.1006/geno.1996.4494.
Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species.
近年来，大鼠作为一种用于基因研究的模式生物，尤其是用于复杂性状的研究，受到了越来越多的关注，但由于缺乏大插入片段基因组文库，进展有限。在此，我们报道了使用pCGS966载体在AB1380宿主中构建的一个用于大鼠基因组的有序酵母人工染色体（YAC）文库及其特征，该文库包含约40,000个克隆。从166个随机选择的克隆中估计平均大小为736 kb；因此该文库提供了基因组10倍的覆盖率，包含单一序列的概率为99.99%。通过荧光原位杂交分析的39个YAC中有8个被发现是嵌合体，表明嵌合克隆的比例约为20 - 30%。该文库被点样到高密度滤膜上，以便通过杂交鉴定YAC克隆，并使用三维方案进行混合以通过PCR筛选。在用于筛选文库的48个探针中，平均发现9.3个阳性克隆，这与计算得出的文库10倍基因组覆盖率一致。这个YAC文库是大鼠的第一个大插入片段基因组文库。它将作为该物种复杂基因组分析的重要新资源提供给广大研究群体。
