Rapid human genomic DNA cloning into mouse artificial chromosome via direct chromosome transfer from human iPSC and CRISPR/Cas9-mediated translocation

被引:3
|
作者
Miyamoto, Hitomaru [1 ]
Kobayashi, Hiroaki [2 ]
Kishima, Nanami [1 ]
Yamazaki, Kyotaro [3 ]
Hamamichi, Shusei [4 ]
Uno, Narumi [5 ]
Abe, Satoshi [4 ]
Hiramuki, Yosuke [4 ]
Kazuki, Kanako [4 ]
Tomizuka, Kazuma [5 ]
Kazuki, Yasuhiro [1 ,2 ,3 ,4 ]
机构
[1] Tottori Univ, Grad Sch Med Sci, Integrated Med Sci, Dept Chromosome Biomed Engn, 86 Nishi Cho, Yonago, Tottori 6838503, Japan
[2] Tottori Univ, Fac Med, Sch Life Sci, Dept Chromosome Biomed Engn, 86 Nishi Cho, Yonago, Tottori 6838503, Japan
[3] Natl Inst Nat Sci, Exploratory Res Ctr Life & Living Syst ExCELLS, Chromosome Engn Res Grp, 5-1 Higashiyama, Okazaki, Aichi 4448787, Japan
[4] Tottori Univ, Chromosome Engn Res Ctr, 86 Nishi Cho, Yonago, Tottori 6838503, Japan
[5] Tokyo Univ Pharm & Life Sci, Fac Life Sci, Lab Bioengn, 1432-1 Horinouchi, Hachioji, Tokyo 1920392, Japan
关键词
PLURIPOTENT STEM-CELLS; MICROCELL-MEDIATED TRANSFER; CHINESE-HAMSTER; GENE; EXPRESSION; CONSTRUCTION; INDUCTION; APOPTOSIS; VECTORS; HYBRIDS;
D O I
10.1093/nar/gkad1218
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes. [Graphical Abstract]
引用
收藏
页码:1498 / 1511
页数:14
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