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.
Highlights
AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of Adenosine monophosphate (AMP) to IMP
AMPD1 is mainly expressed in the skeletal muscle, whereas AMPD2 is widely expressed in non-muscle tissues and AMPD3 is mainly expressed in erythrocytes[3,4]
To study the physiological function of AMPD1, we obtained a strain of Ampd[1] mutant mice from Knockout Mouse programs (KOMP) repository, which was generated by the knockout-first strategy
Summary
AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of AMP to IMP. 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. Bradley and colleagues report a knockout-first strategy to generate mutant alleles for thousands of genes in mice. The tm1d allele (Fig. 1c) is conventional mutation after crossing tm1c allele with Cre transgenic mice This multi-purpose strategy is applied to European Conditional Mouse Mutagenesis (EUCOMM) and the National Institutes of Health Knockout Mouse programs (KOMP) to generate knockout mice, which are available for the researchers world-wide. Polymorphism in AMPD1 gene has been reported to be associated with metabolic myopathy, which is named as AMP deaminase deficiency[8,9]. Our data indicated that insertion of knockout-first cassette into Ampd[1] gene disrupted the expression of neighboring genes up to 2.4 Mb, which may underlie their neonatal death
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