Targeted Genome Modification in Mice Using Zinc-Finger Nucleases
Targeted Genome Modification in Mice Using Zinc-Finger Nucleases
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DOI：
10.1534/genetics.110.117002
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发表时间：
2010-10-01
期刊：
影响因子：
3.3
通讯作者：
Cui, Xiaoxia
中科院分区：
文献类型：
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作者：
Carbery, Iara D.;Ji, Diana;Cui, Xiaoxia
利用小鼠胚胎干细胞的同源重组基因打靶技术对生物医学研究产生了巨大的影响。这项研究提出了一种功能强大的新技术，使用胚胎注射锌指核酸酶（ZFN）在小鼠中进行更有效，更耗时的基因靶向，该酶产生位点特异性双链断裂，通过非同源末端连接途径通过DNA修复导致插入或缺失。在FVB/N和C57 BL/6小鼠中靶向三个单独的基因，多药耐药1a（Mdr 1a）、锯齿状1（Jag 1）和notch同源物3（Notch 3）。注射ZFN导致20-75%的活产婴儿出现一系列特异性基因缺失，从几个核苷酸到长度>1000 bp。修饰的等位基因通过种系有效地传递，并且在短短4个月内获得了针对靶向修饰的纯合动物。此外，该技术可以适应任何遗传背景，消除了世代回交以获得同类动物的需要。我们还验证了Mdr 1a的功能破坏，并证明了ZFN介导的修饰导致真正的敲除。我们的结论是，ZFN技术是一个有效的和方便的替代传统的基因打靶，将大大促进快速创建小鼠模型和功能基因组学研究。
Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.