Pan Yongcheng, Zhang Lusi, Liu Qiong, Li Ying, Guo Hui, Peng Yu, Peng Hexiang, Tang Beisha, Hu Zhengmao, Zhao Jingping, Xia Kun, Li Jia-Da
Key laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China.
The State Key Laboratory of Medical Genetics and School of Life Sciences, Central South University, Changsha, Hunan, China.
Sci Rep. 2016 Oct 24;6:35970. doi: 10.1038/srep35970.
AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of AMP to IMP. To understand the physiological function of AMPD1, we obtained a strain of Ampd1 mutant mice from KOMP repository, which was generated by a knockout-first strategy. An elevated AMP level and almost complete lack of IMP was detected in the skeletal muscle of E18.5 Ampd1 mice. However, Ampd1 mice died in 2 days postnatally, which was contradicting to previous reports. After removal of the knockout-first cassette and critical exon, mice homozygous for the Ampd1 and Ampd1 alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1 mice, but normal in the Ampd1 and Ampd1 mice. The neonatal lethality phenotype in the Ampd1 mice was consistent with the Man1a2-deficient mice. Our results indicated the knockout-first cassette may cause off-target effect by influence the expression of neighboring genes. This study, together with other reports, strongly suggests that removal of targeting cassette by site-specific recombinases is very important for the accurate phenotypic interpretation on mice generated by target mutations.
AMPD1是一种催化AMP脱氨生成IMP的腺苷单磷酸脱氨酶。为了解AMPD1的生理功能，我们从KOMP资源库获得了一株Ampd1突变小鼠，该小鼠是通过敲除优先策略产生的。在E18.5 Ampd1小鼠的骨骼肌中检测到AMP水平升高且几乎完全缺乏IMP。然而，Ampd1小鼠在出生后2天死亡，这与先前的报道相矛盾。去除敲除优先盒和关键外显子后，Ampd1和Ampd1等位基因纯合的小鼠存活至成年。RNA测序分析表明，两个相邻基因Man1a2和Nras的表达在Ampd1小鼠中受到破坏，但在Ampd1和Ampd1小鼠中正常。Ampd1小鼠的新生儿致死表型与Man1a2缺陷小鼠一致。我们的结果表明，敲除优先盒可能通过影响相邻基因的表达而导致脱靶效应。这项研究与其他报道一起强烈表明，通过位点特异性重组酶去除靶向盒对于准确解释由靶向突变产生的小鼠的表型非常重要。
