Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system
Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system
复制标题
DOI：
10.1007/s00335-014-9524-0
复制
发表时间：
2014-08-01
期刊：
影响因子：
2.5
通讯作者：
Yagami, Ken-ichi
中科院分区：
文献类型：
--
作者：
Mizuno, Seiya;Tra Thi Huong Dinh;Yagami, Ken-ichi
单核苷酸突变（SNM）与多种人类疾病有关。CRISPR/Cas9基因组编辑系统有望作为一种遗传修饰方法用于生产SNM诱导的小鼠。为了研究是否可以通过受精卵显微注射CRISPR/Cas9载体和单链DNA（ssDNA）供体来产生SNM诱导的小鼠，我们尝试从有色C57 BL/6 J受精卵产生携带Tyr基因SNM（G291 T）的白化病C57 BL/6 J小鼠。我们首先设计并构建了Tyr基因的CRISPR/Cas9表达载体（px 330-Tyr-M）。px 330-Tyr-M在Tyr基因的靶位点处的DNA切割活性通过EGxxFP系统确认。我们还设计了用于同源定向修复（HDR）介导的基因修饰的ssDNA供体。将px 330-Tyr-M载体和ssDNA供体共显微注射到来自C57 BL/6 J小鼠的224个单细胞期胚胎的原核中。我们获得了60例新生儿，其中28例表现为眼白化病和缺乏被毛色素沉着。对白化病小鼠的基因组测序分析显示，SNM的靶点，Tyr基因中的G291 T，发生在11只小鼠中，并且一只建立者是同源突变的。其余无Tyr G291 T突变的白化病创始人也具有邻近Tyr基因座中靶位点的双等位基因缺失和插入突变体。通过显微注射px 330-Tyr-M DNA载体和突变ssDNA（G291 T in Tyr）供体的C57 BL/6 J受精卵，提供了白化病C57 BL/6 J小鼠的简单生产。CRISPR/Cas9载体和任选的突变体ssDNA的组合有望有效地产生用于研究人类疾病的新型SNM诱导的小鼠模型。
Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.