Caspary Tamara
Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA.
Methods Enzymol. 2010;477:313-27. doi: 10.1016/S0076-6879(10)77016-6.
In the past decade, forward genetic screens in the mouse have come into their own as a practical method for exploring the genetic basis of many biological processes. By looking directly for disruption in a process of interest, genetic screens have always been powerful, but completion of the genome sequence has made mouse forward genetic screens practical, as well. The sequenced genome means we can map and sequence more efficiently than before, so small focused screens are now within the reach of even small labs. N-Ethyl-N-nitrosourea (ENU) is the preferred mutagen in forward genetic screens, because it is extremely potent in the premeiotic male germ line, where it induces point mutations. This last point is crucial, as point mutations lead to all classes of mutations (e.g., null, hypomorphs, neomorphs, antimorphs, and hypermorphs), which is why forward genetic screens can implicate a gene in a particular process when a targeted deletion may not. Point mutations often mimic human disease states, yielding highly relevant animal models. Since mammals reproduce, lactate, behave, develop, and protect themselves from infection differently from other vertebrates, mammalian forward genetic screens are uniquely informative. In fact, in the past decade, forward genetics has uncovered mutations demonstrating that certain genes exist only in mammals, that specific mechanisms function only in mammals, and that particular biological processes may exist only in mammals; hence screens focused on these processes have identified unsuspected genes. As powerful as the approach is, forward genetics remains a method for the committed; the process of screening requires organization and tenacity. This chapter is intended to help those who are ready to make the commitment by providing practical advice. To this end I detail the issues surrounding screen design and screen execution, as well as mutation identification and confirmation.
在过去十年中，小鼠正向遗传学筛选已成为探索许多生物学过程遗传基础的实用方法。通过直接寻找感兴趣过程中的破坏情况，遗传筛选一直都很强大，但基因组序列的完成也使小鼠正向遗传学筛选变得切实可行。已测序的基因组意味着我们能够比以往更高效地进行定位和测序，所以即使是小实验室现在也能开展小型的针对性筛选。N-乙基-N-亚硝基脲（ENU）是正向遗传学筛选中首选的诱变剂，因为它在减数分裂前的雄性生殖系中具有极强的诱变能力，能诱导点突变。最后这一点至关重要，因为点突变可导致所有类型的突变（如无效突变、亚效等位基因、新等位基因、反效等位基因和超效等位基因），这就是为什么当靶向缺失可能无法揭示特定过程中的相关基因时，正向遗传学筛选却能够做到的原因。点突变常常模拟人类疾病状态，从而产生高度相关的动物模型。由于哺乳动物在繁殖、泌乳、行为、发育以及抵御感染等方面与其他脊椎动物存在差异，所以哺乳动物正向遗传学筛选具有独特的信息价值。事实上，在过去十年中，正向遗传学已经发现了一些突变，这些突变表明某些基因仅存在于哺乳动物中，特定机制仅在哺乳动物中发挥作用，特定生物学过程可能仅存在于哺乳动物中；因此，针对这些过程的筛选已经鉴定出了一些未曾预料到的基因。尽管这种方法很强大，但正向遗传学仍然是一种需要坚定决心才能采用的方法；筛选过程需要组织协调和坚韧不拔的精神。本章旨在通过提供实用建议来帮助那些准备好下定决心的人。为此，我详细阐述了筛选设计、筛选实施以及突变鉴定和确认等方面的问题。
