Knöll A, Jacobson D P, Nishino H, Kretz P L, Short J M, Sommer S S
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA.
Mutat Res. 1996 Jun 10;352(1-2):9-22. doi: 10.1016/0027-5107(95)00159-x.
Transgenic animals offer a powerful tool to study the mechanisms of spontaneous and induced mutagenesis in vivo. Herein we used a test version of a growth selectable assay to obtain spontaneous mutants in a lacI target transgene recovered from lacI transgenic B6C3F1 mice (Big Blue). This selection system may have certain advantages relative to the more established plaque screening system for mutation detection because: (1) the plating density of the phage is up to 60 times higher in the selectable assay, reducing the number of plates needed to be screened for a comparable amount of mutants; and (2) the mutant frequency obtained from the selectable assay is higher compared to the plaque assay, possibly due to a higher sensitivity for weaker mutants. However, the longer incubation time of the growth selectable assay might allow E. coli host derived mutants to appear. To address this issue, we investigated the sequence changes in the amino-terminal domain of the lacI gene of 405 mutants derived from the liver, spleen, brain, germ cells and skin of five untreated 6-week-old mice. The mutant colonies were isolated after 60, 84, 108 and 150 h of incubation under growth selectable conditions. Tissue-specific differences in the mutational pattern obtained after 60 and 84 h disappear after a longer time of incubation, possibly due to an increasing contribution of E. coli derived mutants. The evolving selectable systems offer the potential to increase screening efficiency, but the results suggest caution in interpreting data from this system because repair by E. coli of DNA lesions or mismatched heteroduplexes either originating in mouse in vivo or produced by ex vivo manipulation as well as de novo mutations in E. coli might contribute significantly to the observed mutational spectra at each timepoint.
转基因动物为研究体内自发和诱导突变机制提供了强大工具。在此，我们使用了一种生长选择测定的测试版本，以从lacI转基因B6C3F1小鼠（大蓝鼠）中回收的lacI靶基因中获得自发突变体。相对于更成熟的用于突变检测的噬菌斑筛选系统，该选择系统可能具有某些优势，原因如下：（1）在选择测定中噬菌体的铺板密度高达60倍，减少了筛选相当数量突变体所需的平板数量；（2）与噬菌斑测定相比，从选择测定中获得的突变频率更高，这可能是由于对较弱突变体的敏感性更高。然而，生长选择测定较长的孵育时间可能会使大肠杆菌宿主衍生的突变体出现。为了解决这个问题，我们研究了来自五只未经处理的6周龄小鼠的肝脏、脾脏、大脑、生殖细胞和皮肤的405个突变体的lacI基因氨基末端结构域的序列变化。在生长选择条件下孵育60、84、108和150小时后分离出突变菌落。孵育60和84小时后获得的突变模式中的组织特异性差异在更长时间的孵育后消失，这可能是由于大肠杆菌衍生的突变体的贡献增加。不断发展的选择系统有可能提高筛选效率，但结果表明在解释该系统的数据时要谨慎，因为大肠杆菌对源自小鼠体内或体外操作产生的DNA损伤或错配异源双链体的修复以及大肠杆菌中的从头突变可能对每个时间点观察到的突变谱有显著贡献。
