Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.
Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.
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DOI：
10.1007/s11248-015-9884-6
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发表时间：
2015-08
影响因子：
3
通讯作者：
Ohbayashi T
中科院分区：
文献类型：
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作者：
Yoshimura Y;Nakamura K;Endo T;Kajitani N;Kazuki K;Kazuki Y;Kugoh H;Oshimura M;Ohbayashi T
小鼠人工染色体（MAC）作为基因递送载体具有几个优点，包括外源遗传物质的稳定附加型维持和携带大的和/或多个基因插入物（包括其调控元件）的能力。先前，产生了含有多整合位点的MAC（MI-MAC）以促进将多个基因转移到期望的细胞中。为了产生含有具有目的基因的MI-MAC的转染色体（Tc）小鼠，将所需基因插入CHO细胞中的MI-MAC中，然后通过微细胞介导的染色体转移（MMCT）将MI-MAC转移到小鼠胚胎干（mES）细胞中。然而，MMCT从CHO到mES细胞的效率非常低（<10−6）。在这项研究中，我们构建了含有MI-MAC载体的mES细胞系，通过一种简单的转染方法将目的基因直接插入到mES细胞的MI-MAC中，用于Tc小鼠的产生。在MI-MAC mES细胞中，MI-MAC上每个附着位点（FRT、PhiC 31 attP、R4 attP、TP 901 -1attP和Bxb 1attP）处的GFP基因的重组率大于50%。从MI-MAC mES细胞系产生具有高毛色嵌合的嵌合小鼠，并观察到嵌合体的种系传递。作为通过MI-MAC mES方法产生具有所需基因的Tc小鼠的实例，有效地建立了广泛表达Emerald荧光素酶的Tc小鼠品系。因此，研究结果表明，这种采用mES细胞和MI-MAC载体的新Tc策略对于动物转基因是有效和有用的。本文的在线版本（doi：10.1007/s11248-015-9884-6）包含补充材料，可供授权用户使用。
The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10−6). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50 % in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis. The online version of this article (doi:10.1007/s11248-015-9884-6) contains supplementary material, which is available to authorized users.