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

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.

SUBMITTER: Pan Y

PROVIDER: S-EPMC5075929 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Pan Yongcheng Y Zhang Lusi L Liu Qiong Q Li Ying Y Guo Hui H Peng Yu Y Peng Hexiang H Tang Beisha B Hu Zhengmao Z Zhao Jingping J Xia Kun K Li Jia-Da JD

Scientific reports 20161024

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<sup>tm1a/tm1a</sup> mice. However, Ampd1<sup>tm1a/tm1a</sup> mice died in 2 days postnatally, which was contradicting to previous reports. ...[more]

PMID: 27775065

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Project description:We reported previously that adult (HRAS-/-; NRAS-/-) double knockout (DKO) mice showed no obvious external phenotype although lower-than-expected numbers of weaned DKO animals were consistently tallied after crossing NRAS-KO and HRAS-KO mice kept on mixed genetic backgrounds. Using mouse strains kept on pure C57Bl/6 background, here we performed an extensive analysis of the offspring from crosses between HRAS-KO and NRAS-KO mice and uncovered the occurrence of very high rates of perinatal mortality of the resulting DKO littermates due to respiratory failure during the first postnatal 24-48 h. The lungs of newborn DKO mice showed normal organ structure and branching but displayed marked defects of maturation including much-reduced alveolar space with thick separating septa and significant alterations of differentiation of alveolar (AT1, AT2 pneumocytes) and bronchiolar (ciliated, Clara cells) cell lineages. We also observed the retention of significantly increased numbers of undifferentiated progenitor precursor cells in distal lung epithelia and the presence of substantial accumulations of periodic acid-Schiff-positive (PAS+) material and ceramide in the lung airways of newborn DKO mice. Interestingly, antenatal dexamethasone treatment partially mitigated the defective lung maturation phenotypes and extended the lifespan of the DKO animals up to 6 days, but was not sufficient to abrogate lethality in these mice. RNA microarray hybridization analyses of the lungs of dexamethasone-treated and untreated mice uncovered transcriptional changes pointing to functional and metabolic alterations that may be mechanistically relevant for the defective lung phenotypes observed in DKO mice. Our data suggest that delayed alveolar differentiation, altered sphingolipid metabolism and ceramide accumulation are primary contributors to the respiratory stress and neonatal lethality shown by DKO mice and uncover specific, critical roles of HRAS and NRAS for correct lung differentiation that are essential for neonatal survival and cannot be substituted by the remaining KRAS function in this organ.

Dataset Information

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

Publications

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

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