Insertion of a knockout-first cassette in Ampd1 gene leads to neonatal death by disruption of neighboring genes expression

Yongcheng Pan; Lusi Zhang; Qiong Liu; Ying Li; Hui Guo; Yu Peng; Hexiang Peng; Beisha Tang; Zhengmao Hu; Jingping Zhao; Kun Xia; Jia-Da Li

doi:10.1038/srep35970

Insertion of a knockout-first cassette in Ampd1 gene leads to neonatal death by disruption of neighboring genes expression

Sci Rep. 2016 Oct 24:6:35970. doi: 10.1038/srep35970.

Authors

Yongcheng Pan^{1

2}, Lusi Zhang^{3

2}, Qiong Liu², Ying Li², Hui Guo², Yu Peng², Hexiang Peng², Beisha Tang^{1

2

4}, Zhengmao Hu², Jingping Zhao⁵, Kun Xia^{2

6

7}, Jia-Da Li^{1

2

7}

Affiliations

¹ Key laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China.
² The State Key Laboratory of Medical Genetics and School of Life Sciences, Central South University, Changsha, Hunan, China.
³ Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
⁴ Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, China.
⁵ Mental Health Institute of the Second Xiangya Hospital, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, Hunan, China.
⁶ College of Life Science and Technology, Xinjiang University, Xinjiang, China.
⁷ Collaborative Innovation Center for Genetics and Development, Shanghai, China.

Abstract

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^tm1a/tm1a mice. However, Ampd1^tm1a/tm1a 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^tm1c/tm1c and Ampd1^tm1d/tm1d alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1^tm1a/tm1a mice, but normal in the Ampd1^tm1c/tm1c and Ampd1^tm1d/tm1d mice. The neonatal lethality phenotype in the Ampd1^tm1a/tm1a 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

AMP Deaminase / genetics*
Adenosine Monophosphate / analysis
Animals
Animals, Newborn
Gene Expression Regulation*
Gene Knockout Techniques*
Inosine Monophosphate / analysis
Mice
Mice, Knockout
Muscle, Skeletal / pathology
Mutagenesis, Insertional*

Substances

Inosine Monophosphate
Adenosine Monophosphate
AMP Deaminase
AMPD1 protein, mouse