The kick-in system: a novel rapid knock-in strategy.
The kick-in system: a novel rapid knock-in strategy.
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DOI：
10.1371/journal.pone.0088549
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Hirose S
中科院分区：
文献类型：
--
作者：
Tomonoh Y;Deshimaru M;Araki K;Miyazaki Y;Arasaki T;Tanaka Y;Kitamura H;Mori F;Wakabayashi K;Yamashita S;Saito R;Itoh M;Uchida T;Yamada J;Migita K;Ueno S;Kitaura H;Kakita A;Lossin C;Takano Y;Hirose S
敲入鼠模型对我们对人类疾病的理解做出了巨大贡献。然而，一代敲入动物需要投入大量的时间和精力。我们通过开发一种新型的敲入系统来解决这个问题，该系统通过建立具有包裹特定基因组靶点的受体元件的干细胞来绕过几个传统的挑战。一旦建立，这些受体胚胎干细胞(ES)在使用改进的CRE/LOX技术定向整合突变的目标DNA方面是有效的。这是有利的，因为敲入不限于一种先验选择的变体。相反，有可能产生几个突变的动物品系，这些突变动物品系在目标区域具有所需的改变。受体ES细胞的生成是限速步骤，持续约2个月。随后的动物生产操作需要额外的8周时间，但这界定了从基因改变的受孕到动物纳入的整个时间段。我们称这个系统为“启动”系统，以强调其快速和方便的独特特性。为了证明启动方法学的功能，我们产生了两个具有独立突变版本的电压依赖性钾通道Kv7.2(KCNQ2)的小鼠系：p.Tyr284Cys(Y284C)和p.Ala306Thr(A306T)；这两个突变与良性家族性新生儿癫痫有关。Y284C纯合子的成年小鼠出现自发性癫痫发作，而A306T纯合子则过早死亡。两个品系的杂合子小鼠都表现出对戊四氮的敏感性增加，这可能是由于CA1区锥体神经元的M电流减少所致。我们对A306T动物的观察与传统敲入技术获得的结果相匹配，表明踢入系统可以很容易地产生携带各种突变的小鼠，使其成为一种适合简化表型鉴定的饲养技术。
Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a “kick-in” to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.
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