Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes
Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes
复制标题
通过将编码转录激活剂样效应核酸酶 (TALEN) 的 mRNA 注射到受精卵中来产生 Foxo3 靶向小鼠
DOI：
10.1111/rda.12515
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发表时间：
2015
影响因子：
1.7
通讯作者：
石德顺
中科院分区：
文献类型：
--
作者：
朱鹏;刘庆友;刘帅;冯万友;雷小灿;LiuJ;崔奎青;黄奔;石德顺
为探讨FOXO_3参与原始卵母细胞激活调控的机制，将编码转录激活因子样效应子核酸酶(TALEN)的mRNAs注入受精卵中，建立FOXO_3靶向小鼠。TALEN位点是由商业生物公司设计的，在猪、牛、水牛和小鼠之间具有高度保守的同源区。用双荧光素酶报告系统检测人胚胎肾293T(HEK-293T)细胞的TALENS突变非同源末端连接(NHEJ)修复活性为31.3%。然后，我们首先通过显微操作将TALEN-mRNAs注射到小鼠受精卵的细胞质中，测序证实48个囊胚中有4个是突变的。随后，通过原核显微操作技术将TALEN-mRNAs注射到受精卵中，我们获得了20只FVB/NJ背景小鼠的7个Foxo3突变体，它们是Foxo3不依赖的等位基因，具有移码和缺失突变。有趣的是，这7只小鼠都是杂合突变体(foxo3−/+)，其基因突变率达到35%。这5只Foxo3突变雌性在出生后6个月内都不育。组织学检查结果显示，与野生型雌性小鼠相比，Foxo3突变型雌性小鼠卵巢内有稀少的原始卵泡和初级卵泡。此外，两个突变的雄性中有一个是不育的，另一个是正常生育的。这些结果表明，Foxo3的突变在一定程度上严重影响了雌性和雄性的生育能力；此外，一种更有效的基于TALENS的基因突变方法有望为小鼠和其他物种的遗传学研究带来革命性的变化。
ContentsIn this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3‐targeted mice were generated by injection of mRNAs encoding transcription activator‐like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio‐companies. The TALENs mutagenic non‐homologous end‐joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK‐293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN‐mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN‐mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3‐independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3−/+), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide‐type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs‐based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.