Brain-specific PAPP-A knock-out mice?

Abstract

PAPP-A knock-out (KO) mice are a valuable model for investigating the effects of down-regulating localized insulin-like growth factor (IGF) action, which has been shown to extend lifespan and healthspan when the PAPP-A gene is globally deleted. Based on previous mouse models of brain-specific reduction in IGF signaling associated with longevity, we sought to generate brain-specific PAPP-A KO mice and determine effects on metabolism and lifespan. Mice with the PAPP-A gene floxed (fPAPP-A) were crossed with Nestin promoter-driven Cre recombinase transgenic mice. This cross-breeding of mice for Nestin-Cre and mice with other floxed target alleles has been used extensively to investigate brain-specific effects. Our cross-breeding generated four genotypes for study: fPAPP-A/Nestin positive (brain-specific PAPP-A KO); fPAPP-A/Nestin negative (Control for floxed PAPP-A); WT/Nestin positive (Control for Nestin-Cre); WT/Nestin negative (Wild-type Control). The basic genotype screen of neonatal tail snip DNA clearly indicated PAPP-A gene status and the presence (pos) or absence (neg) of Nestin-Cre. We then determined tissue specificity of PAPP-A gene excision. We had expected fPAPP-A/pos mice to be relatively brain-specific for PAPP-A gene deletion and the controls (fPAPP-A/neg, WT/neg and WT/pos mice) to show no effect on PAPP-A expression in brain or other tissues. However, in fPAPP-A/neg mice we found evidence of PAPP-A excision in all tissues examined, i.e., in the presumed absence of Nestin-Cre, indicating germline recombination. We further found that fPAPP-A/pos mice showed near complete excision of the PAPP-A gene in brain, but some also showed germline recombination affecting all tissues tested. To determine if the level of excision indicated by tissue genotyping approximated PAPP-A mRNA expression, we performed RT-qPCR. fPAPP-A/pos mice that showed markedly decreased whole brain PAPP-A mRNA expression (~80%), with little or no effect on expression in the other tissues tested, were designated as "brain-specific" PAPP-A KO. fPAPP-A/pos mice that showed germline recombination had similar decreases in PAPP-A expression in brain but also showed 40-65% decreased PAPP-A mRNA expression in other tissues as well, which was especially striking in kidney, tibia, thymus and spleen. These were designated as "non-specific" PAPP-A KO mice. With unknown and unpredictable specificity until harvest, we chose to assess a surrogate marker of lifespan i.e., thymic involution, in 15- to 18-month-old fPAPP-A/pos and WT/pos mice, the latter an important control for a possible effect of Nestin-Cre per se. Diminished thymic involution as indicated by increased thymic weight (135%, P = 0.035) and decreased histological disruption was seen in "non-specific" PAPP-A KO mice, similar to what was previously reported in 18-month-old global PAPP-A KO mice. There was no significant difference between "brain-specific" PAPP-A KO and control mice. This study highlights the importance of thorough characterization of assumed tissue-specific mouse models and awareness of potential germline recombination for proper data interpretation.

Keywords: Brain; Lifespan; Nestin-Cre; PAPP-A; Thymus.

Figure 1.

Genotyping of tissues from fPAPP-A/neg mice B, brain; L, liver; H, heart; Q, quadriceps; Sp, spleen; K, kidney; MF, mesenteric fat; Tb; tibia; Thy, thymus Arrow indicates the band for excised PAPP-A

Figure 2.

Genotyping of tissues from fPAPP-A/pos mice (a) Example of brain-specific PAPP-A excision (b) Example of non-specific PAPP-A excision – mosaic (c) Example of non-specific PAPP-A excision -- complete B, brain; L, liver; H, heart; Q, quadriceps; Sp, spleen; K, kidney; MF, mesenteric fat; Tb; tibia; Thy, thymus Arrow indicates the band for excised PAPP-A