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.

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 Ampd1tm1a/tm1a mice. However, Ampd1tm1a/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 Ampd1tm1c/tm1c and Ampd1tm1d/tm1d alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1tm1a/tm1a mice, but normal in the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d mice. The neonatal lethality phenotype in the Ampd1tm1a/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