Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition.
Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition.
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DOI：
10.1007/s11248-010-9386-5
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发表时间：
2011-02
影响因子：
3
通讯作者：
Fahrenkrug SC
中科院分区：
文献类型：
--
作者：
Carlson DF;Geurts AM;Garbe JR;Park CW;Rangel-Filho A;O'Grady SM;Jacob HJ;Steer CJ;Largaespada DA;Fahrenkrug SC
对人类疾病相关模型的高度关注增加了对生殖系转基因改进方法的需求。我们描述了通过化学修饰睡美人转座子在创建转基因实验室小鼠和大鼠方面的重大改进。注射前对转座子进行胞嘧啶-磷酸二酯-鸟嘌呤甲基化，可显著增强小鼠和大鼠的种系转基因。与非甲基化转座子相比，甲基化转座子产生的创始小鼠的转基因等位基因遗传率也更高。人工甲基化在早期胚胎中被重新编程，导致表达转基因的创始人。我们还注意到基于甲基化和质粒构象（线性与超螺旋）的影响，转基因插入数量和结构（单插入与串联体）的差异，超螺旋底物导致有效的转座转基因（TnT），几乎消除了串联体插入。综上所述，这些底物修饰增加了转基因建立者的频率和每个建立者的转基因数量，显著提高了潜在转基因品系的数量。由于其简单，多功能性和高效率，增强睡美人成分的TnT代表了一种令人信服的非病毒方法来修饰哺乳动物种系。
Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.
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影响因子：
14.9
作者：
Clark KJ;Carlson DF;Leaver MJ;Foster LK;Fahrenkrug SC
通讯作者：
Fahrenkrug SC
影响因子：
30.8
作者：
Garrick, D;Fiering, S;Whitelaw, E
通讯作者：
Whitelaw, E
影响因子：
56.9
作者：
Giraldez, AJ;Mishima, Y;Schier, AF
通讯作者：
Schier, AF
影响因子：
64.8
作者：
Grimm, Dirk;Streetz, Konrad L.;Kay, Mark A.
通讯作者：
Kay, Mark A.
影响因子：
12.4
作者：
Geurts, AM;Yang, Y;Hackett, PB
通讯作者：
Hackett, PB