Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei 11217, Taiwan.
BMC Biotechnol. 2014 Feb 21;14:16. doi: 10.1186/1472-6750-14-16.
Insertion duplication mutagenesis (IDM) and in-frame deletion (IFD) are common techniques for studying gene function, and have been applied to pneumolysin (ply), a virulence gene in Streptococcus pneumoniae (D39). Discrepancies in virulence between the two techniques were observed in both the previous and present studies. This phenomenon was also observed during mutation analysis of autolysin (lytA).
Our data showed that target gene restoration (TGR) occurred in IDM mutants, even in the presence of antibiotics, while the IFD mutants were stable. In PCR result, TGR occurred later in IDM-ply and -lytA mutants cultured in non-supplemented medium (4-5 h) compared with those grown in medium supplemented with erythromycin (erm)/chloramphenicol (cat) (3-4 h), but plateaued faster. Real-time PCR for detecting TGR had been performed. When compared with 8-h culture, TGR detection increased from Day 1 and Day 2 of IDM mutant's culture. erm-sensitive clones from IDM mutant were found. Southern blot hybridization and Western blotting also confirmed the phenomenon of TGR. The median survival of mice following intraperitoneal (IP) injection with a 3-h culture of IDM-mutants was significantly longer than that with an 8-h culture, irrespective of antibiotic usage. The median survival time of mice following IP injection of a 3-h culture versus an 8-h culture of IDM-ply in the absence of antibiotics was 10 days versus 2 days (p = 0.031), respectively, while in the presence of erm, the median survival was 5 days versus 2.5 days (p = 0.037), respectively. For an IDM-lytA mutant, the corresponding values were 8.5 days versus 2 days (p = 0.019), respectively, for non-supplemented medium, and 2.5 versus 2 days (p = 0.021), respectively, in the presence of cat. A comparable survival rate was observed between WT D39 and an 8-h IDM culture.
TGR in IDM mutants should be monitored to avoid inconsistent results, and misinterpretation of data due to TGR could lead to important biological meaning being overlooked. Therefore, based on these results, IFD is preferable to IDM for disruption of target genes.
插入重复诱变（IDM）和框内缺失（IFD）是研究基因功能的常用技术，已应用于肺炎链球菌（D39）的毒力基因肺炎球菌溶血素（ply）。在先前和目前的研究中，这两种技术的毒力差异观察到不一致。这种现象在自溶素（lytA）的突变分析中也观察到。
我们的数据表明，即使存在抗生素，IDM 突变体中也会发生靶基因恢复（TGR），而 IFD 突变体则稳定。在未添加培养基（4-5 小时）中培养的 IDM-ply 和 -lytA 突变体的 PCR 结果中，TGR 发生的时间晚于添加红霉素（erm）/氯霉素（cat）的培养基（3-4 小时），但更快达到平台期。已经进行了用于检测 TGR 的实时 PCR。与 8 小时培养相比，IDM 突变体培养的第 1 天和第 2 天检测到 TGR 增加。从 IDM 突变体的 erm 敏感克隆中发现了 TGR。Southern 印迹杂交和 Western 印迹也证实了 TGR 的现象。无论是否使用抗生素，经腹腔（IP）注射 3 小时 IDM 突变体培养物的小鼠中位存活时间明显长于注射 8 小时培养物的小鼠。在不使用抗生素的情况下，经 IP 注射 3 小时 IDM-ply 培养物与 8 小时培养物的小鼠中位生存时间分别为 10 天与 2 天（p=0.031），而在用 erm 时，中位生存时间分别为 5 天与 2.5 天（p=0.037）。对于 IDM-lytA 突变体，在不添加培养基的情况下，相应的值分别为 8.5 天与 2 天（p=0.019），而在添加 cat 的情况下，相应的值分别为 2.5 天与 2 天（p=0.021）。WT D39 与 8 小时 IDM 培养物的存活率相当。
应监测 IDM 突变体中的 TGR，以避免因 TGR 导致结果不一致和数据误读，因为 TGR 可能导致重要的生物学意义被忽视。因此，基于这些结果，IFD 优于 IDM 用于靶基因的破坏。
