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 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.

Reevaluation of the Role of ERK3 in Perinatal Survival and Post-Natal Growth Using New Genetically-Engineered Mouse Models

“…However, an accumulating number of reports indicate that some phenotypes associated with a targeted gene mutation are causally related to off-target effects on neighboring genes (17-21). For example, mice with a homozygous disruption of the Ampd1 gene generated by a targeted trap mutation (knockout-first allele) die within two days post-natally, and show reduced body weight, decreased lung saccular space and lack of milk in their stomach (20). However, after removal of the knockout-first cassette containing the lacz and neo r genes to generate a conditional allele and further deletion of exon 3 of the Ampd1 gene to produce a null allele, homozygous mutant mice survived to adulthood.…”

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan

“…S1a). Presence of pgk-Neo cassette either in the intron or the 3′UTR is known to cause a position dependent long-range disruption of endogenous gene expression (Pham et al 1996, Seidl et al 1999, Strathdee et al; 2008, Lou et al 2009; Pan et al 2016). …”

“…The constitutively active pgk promoter that transcribes neomycin gene is placed in an opposite orientation to the endogenous Acan gene promoter (Supporting Information Figure S1a). Presence of pgk-Neo cassette either in the intron or the 3 0 UTR is known to cause a position dependent long-range disruption of endogenous gene expression (Lou et al, 2009;Pan et al, 2016;Pham, MacIvor, Hug, Heusel, & Ley 1996;Seidl et al 1999;Strathdee, Whitelaw, & Clark, 2008).…”

Reevaluation of the Role of Extracellular Signal-Regulated Kinase 3 in Perinatal Survival and Postnatal Growth Using New Genetically Engineered Mouse Models

“…The impact of local changes in gene expression on the phenotypic spectrum of targeted mouse mutants has not been thoroughly studied. However, an accumulating number of reports indicate that some phenotypes associated with a targeted gene mutation are causally related to off-target effects on neighboring genes (17)(18)(19)(20)(21). For example, mice with a homozygous disruption of the Ampd1 gene generated by a targeted trap mutation (knockout-first allele) die within 2 days postnatally and show reduced body weight, decreased lung saccular space, and lack of milk in their stomach (20).…”

“…However, an accumulating number of reports indicate that some phenotypes associated with a targeted gene mutation are causally related to off-target effects on neighboring genes (17)(18)(19)(20)(21). For example, mice with a homozygous disruption of the Ampd1 gene generated by a targeted trap mutation (knockout-first allele) die within 2 days postnatally and show reduced body weight, decreased lung saccular space, and lack of milk in their stomach (20). However, after removal of the knockout-first cassette containing the lacz and neo r genes to generate a conditional allele and further deletion of exon 3 of the Ampd1 gene to produce a null allele, homozygous mutant mice survived to adulthood.…”