Department of Laboratory Animal Science, School of Basic Medical Science, Capital Medical University, Beijing, 100069, China.
Mol Genet Genomics. 2013 Aug;288(7-8):309-16. doi: 10.1007/s00438-013-0751-y. Epub 2013 May 23.
Microsatellites are 50-200 repetitive DNA sequences composed of 1- to 6-base-pair-long reiterative motifs within the genome. They are vulnerable to DNA modifications, such as recombination and/or integration, and are recognized as "sentinel" DNA. Our previous report indicated that the genotypes of the microsatellite loci could change from mono- to poly-morphisms (CMP) in gene knockout (KO) mice, implying that genetic modification induces microsatellite mutation. However, it is still unclear whether the random insertion of DNA fragments into mice genomes produced via transgene (Tg) or N-ethyl-N-nitrosourea (ENU) would also result in microsatellite mutations or microsatellite loci genotypes changes. This study was designed to find possible clues to answer this question. In brief, 198 microsatellite loci that were distributed among almost all of the chromosomes (except for the Y) were examined through polymerase chain reaction to screen possible CMPs in six Tg strains. First, for each strain, the microsatellite sequences of all loci were compared between Tg and the corresponding background strain to exclude genetic interference. Simultaneously, to exclude spontaneous mutation-related CMPs that might exist in the examined six strains, mice from five spontaneously mutated inbred strains were used as the negative controls. Additionally, the sequences of all loci in these spontaneous mutated mice were compared to corresponding genetic background controls. The results showed that 40 of the 198 (20.2%) loci were identified as having CMPs in the examined Tg mice strains. The CMP genotypes were either homozygous or heterozygous compared to the background controls. Next, we applied the 40 CMP positive loci in ENU-mutated mice and their corresponding background controls. After that, a general comparison of CMPs that exist among Tg, ENU-treated and KO mouse strains was performed. The results indicated that four (D11mit258, D13mit3, D14mit102 and DXmit172) of the 40 (10%) CMP loci were shared by Tg and KO mice, two (D15mit5 and D14mit102) (5%) by Tg and ENU-treated mice, and one (D14mit102) (2.5%) by all three genetic modifications. Collectively, our study implies that genetic modifications by KO, Tg or chemical mutant can trigger microsatellite CMPs in inbred mouse strains. These shared microsatellite loci could be regarded as "hot spots" of microsatellite mutation for genetic monitoring in genetic modified mice.
微卫星是基因组内由 1 到 6 个碱基对组成的重复 DNA 序列，长度为 50-200 个碱基对。它们易受 DNA 修饰的影响，如重组和/或整合，并被认为是“哨兵”DNA。我们之前的报告表明，微卫星基因座的基因型可以从单态变为多态（CMP）在基因敲除（KO）小鼠中，这意味着遗传修饰诱导微卫星突变。然而，目前尚不清楚通过转基因（Tg）或 N-乙基-N-亚硝脲（ENU）将 DNA 片段随机插入小鼠基因组是否也会导致微卫星突变或微卫星基因座基因型改变。本研究旨在寻找可能的线索来回答这个问题。简而言之，通过聚合酶链反应（PCR）检查了分布在几乎所有染色体（除了 Y 染色体）上的 198 个微卫星基因座，以筛选 6 个 Tg 株系中可能存在的 CMP。首先，对于每个株系，将 Tg 和相应背景株系之间所有基因座的微卫星序列进行比较，以排除遗传干扰。同时，为了排除可能存在于被检查的 6 个株系中的自发突变相关的 CMP，使用来自 5 个自发突变近交系的小鼠作为阴性对照。此外，将这些自发突变小鼠的所有基因座序列与相应的遗传背景对照进行比较。结果表明，在被检查的 Tg 小鼠株系中，有 40 个（20.2%）基因座被鉴定为存在 CMP。与背景对照相比，CMP 基因型为纯合或杂合。接下来，我们将 40 个 CMP 阳性基因座应用于 ENU 突变小鼠及其相应的背景对照。之后，对 Tg、ENU 处理和 KO 小鼠株系之间存在的 CMP 进行了一般比较。结果表明，在 Tg 和 KO 小鼠中，有 4 个（D11mit258、D13mit3、D14mit102 和 DXmit172）CMP 基因座（10%）是共有的，在 Tg 和 ENU 处理的小鼠中，有 2 个（D15mit5 和 D14mit102）（5%）是共有的，在所有三种遗传修饰中，有 1 个（D14mit102）（2.5%）是共有的。总的来说，我们的研究表明，KO、Tg 或化学诱变引起的遗传修饰可以在近交系小鼠中引发微卫星 CMP。这些共有的微卫星基因座可以被视为遗传修饰小鼠遗传监测的微卫星突变“热点”。
