Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells.
Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells.
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DOI：
10.1093/nar/gks262
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发表时间：
2012-07
影响因子：
14.9
通讯作者：
Ishida Y
中科院分区：
文献类型：
--
作者：
Mayasari NI;Mukougawa K;Shigeoka T;Kawakami K;Kawaichi M;Ishida Y
在目前国际基因敲除小鼠计划（KOMP）中用于灭活胚胎干细胞（ES细胞）中所有小鼠基因的插入诱变技术中，随机基因捕获一直发挥着主要作用。还进行了基因靶向实验，以单独和有条件地敲除剩余的“难以捕获”的基因。在这里，我们表明，在ES细胞中的转录沉默基因是严重不足的随机捕获的基因在KOMP。我们的具有共同逆转录病毒骨架的条件性聚腺苷酸捕获载体也具有被整合到组成型转录基因组位点中的强烈偏好。最重要的是，条件性基因破坏不能成功地通过使用逆转录病毒载体，因为频繁的发展载体内的缺失/重排。我们发现，剪切和粘贴型DNA转座子之一，Tol 2，可以作为一个理想的平台，基因陷阱载体，确保识别和条件性破坏ES细胞中的广谱基因。我们还解决了一个长期存在的问题，与多个载体整合到一个单一的细胞的基因组中，通过纳入差异标记的Tol 2转座子的混合物。我们相信，我们的策略表明了一种直接的方法来大规模生产靶细胞中基因的条件破坏等位基因。
Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually and conditionally knockout the remaining ‘difficult-to-trap’ genes. Here, we show that transcriptionally silent genes in ES cells are severely underrepresented among the randomly trapped genes in KOMP. Our conditional poly(A)-trapping vector with a common retroviral backbone also has a strong bias to be integrated into constitutively transcribed genome loci. Most importantly, conditional gene disruption could not be successfully accomplished by using the retrovirus vector because of the frequent development of intra-vector deletions/rearrangements. We found that one of the cut and paste-type DNA transposons, Tol2, can serve as an ideal platform for gene-trap vectors that ensures identification and conditional disruption of a broad spectrum of genes in ES cells. We also solved a long-standing problem associated with multiple vector integration into the genome of a single cell by incorporating a mixture of differentially tagged Tol2 transposons. We believe our strategy indicates a straightforward approach to mass-production of conditionally disrupted alleles for genes in the target cells.
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影响因子：
56.9
作者：
Carninci, P;Kasukawa, T;Hayashizaki, Y
通讯作者：
Hayashizaki, Y
DOI：
10.1073/pnas.97.21.11403
发表时间：
2000-10-10
影响因子：
11.1
作者：
Kawakami, K;Shima, A;Kawakami, N
通讯作者：
Kawakami, N
影响因子：
46.9
作者：
Buchholz, F;Angrand, PO;Stewart, AF
通讯作者：
Stewart, AF
影响因子：
48
作者：
Ivics, Zoltan;Li, Meng Amy;Mates, Lajos;Boeke, Jef D.;Nagy, Andras;Bradley, Allan;Izsvak, Zsuzsanna
通讯作者：
Izsvak, Zsuzsanna
影响因子：
56.9
作者：
GOSSLER, A;JOYNER, AL;SKARNES, WC
通讯作者：
SKARNES, WC