Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease
Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease
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DOI：
10.1371/journal.pgen.1004951
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发表时间：
2015-02-01
期刊：
影响因子：
4.5
通讯作者：
Grosveld, Gerard C.
中科院分区：
文献类型：
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作者：
Lagutina, Irina V.;Valentine, Virginia;Grosveld, Gerard C.
许多癌症中反复发生的染色体易位导致融合基因的产生，这些融合基因直接涉及致瘤过程。由于融合基因的构建通常完全或部分缺乏正确的调控序列，对小鼠癌症融合基因影响的精确建模一直是不准确的。人肺泡横纹肌肉瘤（a - rms）的互反t(2;13) （q36.1;q14.1）产生了一个典型的PAX3-FOXO1融合基因。在小鼠体内模拟这种易位是复杂的，因为Pax3和Foxo1在各自的染色体上处于相反的方向，这阻碍了通过简单易位形成功能性Pax3-Foxo1融合。为了解决这个问题，我们利用cre介导的位于合成区边缘的两对不相关的反向LoxP位点的重组，不可逆地反转了3号染色体上包含Foxo1的4.9 Mb合成片段的方向。我们测试了纯合小鼠成肌细胞中Pax3和Foxo1基因座的空间接近是否在诱导靶向CRISPR-Cas9核酸酶诱导Pax3和Foxo1 DNA双链断裂时促进了Pax3-Foxo1融合基因的形成。荧光原位杂交表明，前肢成肌细胞Pax3/Foxo1共定位的频率高于后肢成肌细胞。确实，在前肢成肌细胞中通过互反t（1; 3）产生了更多的融合基因，这些基因表达了正确拼接的Pax3-Foxo1 mRNA，编码Pax3-Foxo1融合蛋白。我们得出结论，基因座邻近促进了DNA双链断裂诱导下的染色体易位。鉴于Pax3-Foxo1融合基因将包含精确调控其表达所需的所有调控序列，我们提出CRISPR-Cas9为忠实地模拟小鼠染色体易位引起的人类疾病提供了一种新的手段。
Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13) (q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1; 3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.