Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis.
Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis.
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DOI：
10.1093/nar/gky183
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发表时间：
2018-06-01
影响因子：
14.9
通讯作者：
Takeda J
中科院分区：
文献类型：
--
作者：
Yamanishi A;Matsuba A;Kondo R;Akamatsu R;Tanaka S;Tokunaga M;Horie K;Kokubu C;Ishida Y;Takeda J
单倍体小鼠胚胎干细胞（ESCs），其单个命中突变足以产生功能丧失表型，为前向遗传筛查提供了强大的工具。然而，这种策略可能会受到单倍体ESCs不希望的自身二倍体化的阻碍。为了克服这一障碍，我们设计了一种新的方法，促进在单倍体基因诱变过程中自二倍体化产生的纯合突变体ESC克隆的富集。单倍体小鼠ESCs通过荧光活化细胞分选纯化以保持其单倍体特性，然后用基于Tol2转座子的双等位多a捕获（BPATrap）载体转染，该载体携带可逆G418 +嘌呤霉素双选择盒。G418加嘌呤霉素双重选择富集了双等位突变克隆，这些克隆在单载体插入单倍体基因组后经历了自二倍体化。利用该方法，我们从2208个克隆中剔除杂合子ESCs和多个载体插入的ESCs，成功获得222个纯合子ESCs突变体。在转染Tol2 BPATrap后，单倍体ESCs的细胞分选部分克服了产生纯合突变ESCs的效率相对较低的问题。这些结果表明，我们的方法为胚胎干细胞的诱变和哺乳动物基因组的功能分析提供了一个有效的平台。
Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.
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影响因子：
64.8
作者：
Elling U;Wimmer RA;Leibbrandt A;Burkard T;Michlits G;Leopoldi A;Micheler T;Abdeen D;Zhuk S;Aspalter IM;Handl C;Liebergesell J;Hubmann M;Husa AM;Kinzer M;Schuller N;Wetzel E;van de Loo N;Martinez JAZ;Estoppey D;Riedl R;Yang F;Fu B;Dechat T;Ivics Z;Agu CA;Bell O;Blaas D;Gerhardt H;Hoepfner D;Stark A;Penninger JM
通讯作者：
Penninger JM
影响因子：
14.9
作者：
Mayasari NI;Mukougawa K;Shigeoka T;Kawakami K;Kawaichi M;Ishida Y
通讯作者：
Ishida Y
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
DOI：
10.1073/pnas.0810380105
发表时间：
2008-12-16
影响因子：
11.1
作者：
Urasaki, Akihiro;Asakawa, Kazuhide;Kawakami, Koichi
通讯作者：
Kawakami, Koichi
DOI：
10.1126/science.aad5227
发表时间：
2016-01-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Slaymaker IM;Gao L;Zetsche B;Scott DA;Yan WX;Zhang F
通讯作者：
Zhang F