PDF(Pubmed)
Abstract：
Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.
摘要：
与小鼠模型相比，大鼠动物模型具有相对优越的认知能力和与人体生理特征更高的相似性，因此被广泛使用。然而,由于难以建立胚胎干细胞和进行遗传修饰，它们的使用受到限制，胚胎学研究不足。在这项研究中,我们建立了最佳的超数排卵和受精卵转移条件，包括最佳激素注射浓度（≥150IU/kg的PMSG和hCG）和培养基（mR1ECM），获得高质量的受精卵，建立大鼠体外受精条件。接下来,通过进行PCR分析和T7E1测定选择具有最佳靶向活性的sgRNA，并使用CRISPR/Cas9系统通过诱导fukutin基因缺乏来构建肌营养不良大鼠模型，而没有检测到任何脱靶效应。通过观察体重下降至对照组体重的三分之一，在表型上证实了fukutin敲除大鼠的生产。总之,我们成功地使用CRISPR/CAS9系统构建了第一个肌营养不良症大鼠模型，以增加产生各种动物疾病模型的未来前景，并鼓励使用大鼠进行疾病研究。
