Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.
Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.
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DOI：
10.1155/2017/6037159
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发表时间：
2017
影响因子：
--
通讯作者：
Nonaka K
中科院分区：
文献类型：
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作者：
Sato H;Kato H;Yamaza H;Masuda K;Nguyen HT;Pham TT;Han X;Hirofuji Y;Nonaka K
胚胎三体性会导致人类流产或先天性遗传疾病。最常见的常染色体异常是 13、18 和 21 号染色体三体性。尽管基因剂量的改变被认为会导致染色体额外拷贝引起的疾病，但与特定疾病表型相关的基因仍不清楚。为了从三体细胞生成正常细胞作为病因分析或三体综合征候选疗法的手段，我们开发了一种系统来消除人类细胞中的目标染色体。通过在含有 3 个 21 号染色体拷贝的 HeLa 细胞中整合 DNA 盒，以 21 号染色体为目标。该 DNA 盒包含两个反向 loxP 位点和一个单纯疱疹病毒胸苷激酶 (HSV-tk) 基因。该系统在 Cre 重组酶表达后导致 21 号染色体错误分离，随后通过在包含更昔洛韦 (GCV) 的培养基中培养来选择缺乏染色体的细胞。通过转染 Cre 表达载体，可以有效诱导仅含有两个 21 号染色体拷贝的细胞，表明该方法可用于消除目标染色体。
Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.