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.

PAPP-A 敲除 (KO) 小鼠是研究下调局部胰岛素样生长因子 (IGF) 作用的重要模型，当 PAPP-A 基因在全球范围内时，IGF 已被证明可以延长寿命和健康寿命删除。基于先前与长寿相关的大脑特异性 IGF 信号减少的小鼠模型，我们试图生成大脑特异性 PAPP-A KO 小鼠并确定对新陈代谢和寿命的影响。将 PAPP-A 基因 floxed (fPAPP-A) 的小鼠与巢蛋白启动子驱动的 Cre 重组酶转基因小鼠杂交。这种针对 Nestin-Cre 的小鼠和具有其他 floxed 靶等位基因的小鼠的杂交已被广泛用于研究大脑特异性效应。我们的杂交产生了四种基因型供研究：fPAPP-A/Nestin 阳性（脑特异性 PAPP-A KO）；fPAPP-A/Nestin 阴性（floxed PAPP-A 的对照）；WT/Nestin 阳性（Nestin-Cre 对照）；WT/巢蛋白阴性（野生型对照）。

新生儿剪尾 DNA 的基本基因型筛选清楚地表明了 PAPP-A 基因状态和 Nestin-Cre 的存在 (pos) 或不存在 (neg)。然后我们确定了 PAPP-A 基因切除的组织特异性。我们曾预计 fPAPP-A/pos 小鼠对 PAPP-A 基因缺失具有相对脑特异性，而对照组（fPAPP-A/neg、WT/neg 和 WT/pos 小鼠）对 PAPP-A 表达没有影响脑或其他组织。然而，在 fPAPP-A/neg 小鼠中，我们发现在所有检查的组织中都存在 PAPP-A 切除的证据，即在假定不存在 Nestin-Cre 的情况下，表明种系重组。我们进一步发现，fPAPP-A/pos 小鼠的大脑中 PAPP-A 基因几乎完全切除，但有些还显示出种系重组影响所有测试的组织。

为了确定组织基因分型指示的切除水平是否接近 PAPP-A mRNA 表达，我们进行了 RT-qPCR。fPAPP-A/pos 小鼠表现出全脑 PAPP-A mRNA 表达显着降低 (~80%)，对测试的其他组织中的表达几乎没有影响，被指定为“脑特异性”PAPP-A KO。表现出种系重组的 fPAPP-A/pos 小鼠在脑中 PAPP-A 表达也有类似的下降，但在其他组织中 PAPP-A mRNA 表达也下降了 40-65%，这在肾脏、胫骨、胸腺和脾。这些被指定为“非特异性”PAPP-A KO 小鼠。

在收获前具有未知和不可预测的特异性，我们选择在 15 至 18 个月大的 fPAPP-A/pos 和 WT/pos 小鼠中评估寿命的替代标志物，即胸腺退化，后者是可能的重要控制Nestin-Cre 本身的作用。在“非特异性”PAPP-A KO 小鼠中观察到胸腺退化减少，表现为胸腺重量增加（135%，P  = 0.035）和组织学破坏减少，类似于之前在 18 个月大的全球 PAPP-一只KO老鼠。“大脑特异性” PAPP-A KO 和对照小鼠之间没有显着差异。

本研究强调了对假定的组织特异性小鼠模型进行彻底表征的重要性，以及对潜在种系重组的认识以正确解释数据的重要性。