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

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.

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