Jansa Petr, Divina Petr, Forejt Jirí
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Center for Applied Genomics, Vídenská 1083, CZ-142 20, Prague 4, Czech Republic.
BMC Genomics. 2005 Nov 16;6:161. doi: 10.1186/1471-2164-6-161.
The genome of classical laboratory strains of mice is an artificial mosaic of genomes originated from several mouse subspecies with predominant representation (>90%) of the Mus m. domesticus component. Mice of another subspecies, East European/Asian Mus m. musculus, can interbreed with the classical laboratory strains to generate hybrids with unprecedented phenotypic and genotypic variations. To study these variations in depth we prepared the first genomic large insert BAC library from an inbred strain derived purely from the Mus m. musculus-subspecies. The library will be used to seek and characterize genomic sequences controlling specific monogenic and polygenic complex traits, including modifiers of dominant and recessive mutations.
A representative mouse genomic BAC library was derived from a female mouse of the PWD/Ph inbred strain of Mus m. musculus subspecies. The library consists of 144,768 primary clones from which 97% contain an insert of 120 kb average size. The library represents an equivalent of 6.7 x mouse haploid genome, as estimated from the total number of clones carrying genomic DNA inserts and from the average insert size. The clones were arrayed in duplicates onto eight high-density membranes that were screened with seven single-copy gene probes. The individual probes identified four to eleven positive clones, corresponding to 6.9-fold coverage of the mouse genome. Eighty-seven BAC-ends of PWD/Ph clones were sequenced, edited, and aligned with mouse C57BL/6J (B6) genome. Seventy-three BAC-ends displayed unique hits on B6 genome and their alignment revealed 0.92 single nucleotide polymorphisms (SNPs) per 100 bp. Insertions and deletions represented 0.3% of the BAC end sequences.
Analysis of the novel genomic library for the PWD/Ph inbred strain demonstrated coverage of almost seven mouse genome equivalents and a capability to recover clones for specific regions of PWD/Ph genome. The single nucleotide polymorphism between the strains PWD/Ph and C57BL/6J was 0.92/100 bp, a value significantly higher than between classical laboratory strains. The library will serve as a resource for dissecting the phenotypic and genotypic variations between mice of the Mus m. musculus subspecies and classical laboratory mouse strains.
经典实验室小鼠品系的基因组是一个人工拼接的基因组嵌合体，其起源于几个小鼠亚种，其中小家鼠（Mus m. domesticus）成分占主导（>90%）。另一个亚种，东欧/亚洲小家鼠（Mus m. musculus），可以与经典实验室品系杂交，产生具有前所未有的表型和基因型变异的杂种。为了深入研究这些变异，我们从一个纯源自小家鼠亚种的近交系制备了第一个基因组大插入片段细菌人工染色体（BAC）文库。该文库将用于寻找和表征控制特定单基因和多基因复杂性状的基因组序列，包括显性和隐性突变的修饰基因。
一个具有代表性的小鼠基因组BAC文库源自小家鼠亚种PWD/Ph近交系的一只雌性小鼠。该文库由144,768个原始克隆组成，其中97%包含平均大小为120 kb的插入片段。根据携带基因组DNA插入片段的克隆总数和平均插入片段大小估计，该文库相当于6.7倍的小鼠单倍体基因组。这些克隆被一式两份排列在八张高密度膜上，并用七个单拷贝基因探针进行筛选。各个探针鉴定出4至11个阳性克隆，相当于对小鼠基因组进行了6.9倍的覆盖。对PWD/Ph克隆的87个BAC末端进行了测序、编辑，并与小鼠C57BL/6J（B6）基因组进行比对。73个BAC末端在B6基因组上显示出独特的匹配，它们的比对结果显示每100 bp有0.92个单核苷酸多态性（SNP）。插入和缺失占BAC末端序列的0.3%。
对PWD/Ph近交系新基因组文库的分析表明，其覆盖了近七个小鼠基因组当量，并能够回收PWD/Ph基因组特定区域的克隆。PWD/Ph品系与C57BL/6J品系之间的单核苷酸多态性为0.92/100 bp，这一数值显著高于经典实验室品系之间的数值。该文库将作为一种资源，用于剖析小家鼠亚种小鼠与经典实验室小鼠品系之间的表型和基因型变异。
