Schimenti J C, Libby B J, Bergstrom R A, Wilson L A, Naf D, Tarantino L M, Alavizadeh A, Lengeling A, Bucan M
The Jackson Laboratory, Bar Harbor, Maine 04609 USA.
Genome Res. 2000 Jul;10(7):1043-50. doi: 10.1101/gr.10.7.1043.
Chromosome deletions have several applications in the genetic analysis of complex organisms. They can be used as reagents in region-directed mutagenesis, for mapping of simple or complex traits, or to identify biological consequences of segmental haploidy, the latter being relevant to human contiguous gene syndromes and imprinting. We have generated three deletion complexes in ES (Embryonic Stem) cells that collectively span approximately 40 cM of proximal mouse chromosome 5. The deletion complexes were produced by irradiation of F(1) hybrid ES cells containing herpes simplex virus thymidine kinase genes (tk) integrated at the Dpp6, Hdh (Huntington disease locus), or Gabrb1 loci, followed by selection for tk-deficient clones. Deletions centered at the adjacent Hdh and Dpp6 loci ranged up to approximately 20 cM or more in length and overlapped in an interdigitated fashion. However, the interval between Hdh and Gabrb1 appeared to contain a locus haploinsufficient for ES cell viability, thereby preventing deletions of either complex from overlapping. In some cases, the deletions resolved the order of markers that were previously genetically inseparable. A subset of the ES cell-bearing deletions was injected into blastocysts to generate germline chimeras and establish lines of mice segregating the deletion chromosomes. At least 11 of the 26 lines injected were capable of producing germline chimeras. In general, those that failed to undergo germline transmission bore deletions larger than the germline-competent clones, suggesting that certain regions of chromosome 5 contain haploinsufficient developmental genes, and/or that overall embryonic viability is cumulatively decreased as more genes are rendered hemizygous. Mice bearing deletions presumably spanning the semidominant hammertoe locus (Hm) had no phenotype, suggesting that the classic allele is a dominant, gain-of-function mutation. Overlapping deletion complexes generated in the fashion described in this report will be useful as multipurpose genetic tools and in systematic functional mapping of the mouse genome.
染色体缺失在复杂生物体的遗传分析中有多种应用。它们可用作区域定向诱变的试剂，用于简单或复杂性状的定位，或用于确定部分单倍体的生物学后果，后者与人类相邻基因综合征和印记有关。我们在胚胎干细胞（ES细胞）中产生了三个缺失复合体，它们共同覆盖了小鼠近端5号染色体约40厘摩的区域。这些缺失复合体是通过对含有整合在Dpp6、Hdh（亨廷顿病位点）或Gabrb1位点的单纯疱疹病毒胸苷激酶基因（tk）的F(1)杂交ES细胞进行辐射，然后选择tk缺陷克隆而产生的。以相邻的Hdh和Dpp6位点为中心的缺失长度可达约20厘摩或更长，并以相互交错的方式重叠。然而，Hdh和Gabrb1之间的区间似乎包含一个对ES细胞活力单倍体不足的位点，从而阻止了任何一个复合体的缺失发生重叠。在某些情况下，这些缺失解决了以前在遗传上无法区分的标记顺序。将携带缺失的ES细胞亚群注射到囊胚中，以产生种系嵌合体并建立分离缺失染色体的小鼠品系。注射的26个品系中至少有11个能够产生种系嵌合体。一般来说，那些未能进行种系传递的品系所携带的缺失比具有种系能力的克隆更大，这表明5号染色体的某些区域包含单倍体不足的发育基因，和/或随着更多基因变为半合子，整体胚胎活力会累积下降。携带可能跨越半显性槌状趾位点（Hm）的缺失的小鼠没有表型，这表明经典等位基因是一个显性的功能获得性突变。以本报告中描述的方式产生的重叠缺失复合体将作为多用途遗传工具以及在小鼠基因组的系统功能定位中有用。
