Presence of a Neomycin selection cassette in the Amigo1 locus alters gene expression in the olfactory epithelium leading to region-specific defects in olfactory receptor neuron development

During development of the nervous system, neurons connect to one another in a precisely organized manner. Sensory systems provide a good example of this organization, whereby the composition of the outside world is represented in the brain by neuronal maps. Establishing correct patterns of neural circuitry is crucial, as inaccurate map formation can lead to severe disruptions in sensory processing. In rodents, olfactory stimuli modulate a wide variety of behaviours essential for survival. The formation of the olfactory glomerular map is dependent on molecular cues that guide olfactory receptor neuron axons to broad regions of the olfactory bulb and on cell adhesion molecules that promote axonal sorting into specific synaptic units in this structure. Here, we demonstrate that the cell adhesion molecule Amigo1 is expressed in a subpopulation of olfactory receptor neurons, and we investigate its role in the precise targeting of olfactory receptor neuron axons to the olfactory bulb using a genetic loss-of-function approach in mice. While ablation of Amigo1 did not lead to alterations in olfactory sensory neuron axonal targeting, our experiments revealed that the presence of a neomycin resistance selection cassette in the Amigo1 locus can lead to off-target effects that are not due to loss of Amigo1 expression, including unexpected altered gene expression in olfactory receptor neurons and reduced size of glomeruli in the ventral region of the olfactory bulb. Our results demonstrate that insertion of a neomycin selection cassette into the mouse genome can have specific deleterious effects on the development of the olfactory system and highlight the importance of removing antibiotic resistance cassettes from genetic loss-of-function mouse models when studying olfactory system development. Whole Olfactory Epithelium was dissected from postnatal day 7 mice from three different genotypes for a total of seven samples. One sample was from a wild-type mouse. 3 samples were from Amigo1-/- mice. 3 samples were from Amigo1neo/neo mice (these mice have an insertion of a neomycin cassette in the Amigo1 locus, replacing the Amigo1 coding region)在神经系统发育过程中，神经元彼此间以高度精准的有序方式建立连接。感觉系统便是这种组织模式的绝佳范例：外界环境的构成信息会通过神经元图谱（neuronal maps）在大脑中得以表征。构建正确的神经环路模式至关重要，因为图谱构建失误可导致感觉加工过程出现严重紊乱。 在啮齿类动物中，嗅觉刺激可调控诸多关乎生存的核心行为。嗅小球图谱（olfactory glomerular map）的形成依赖两类关键要素：一是引导嗅觉受体神经元轴突抵达嗅球宽泛区域的分子信号，二是促进轴突在该结构内分选至特定突触单元的细胞黏附分子。 本研究证实，细胞黏附分子Amigo1在部分嗅觉受体神经元中表达，并通过小鼠遗传功能缺失（loss-of-function）实验方法，探究其在嗅觉受体神经元轴突精准靶向嗅球过程中的作用。实验结果显示，敲除Amigo1并未引发嗅觉感觉神经元轴突靶向的异常，但研究团队发现，在Amigo1基因座（locus）中插入新霉素抗性筛选盒（neomycin resistance selection cassette）会产生非因Amigo1表达缺失导致的脱靶效应，包括嗅觉受体神经元中出现意外的基因表达改变，以及嗅球腹侧区域的嗅小球体积缩小。 本研究结果表明，在小鼠基因组中插入新霉素筛选盒会对嗅觉系统发育产生特定的有害影响，并强调了在开展嗅觉系统发育相关研究时，需从遗传功能缺失小鼠模型中移除抗生素抗性筛选盒的重要性。 本研究从3种不同基因型的出生后第7天小鼠体内剥离完整嗅上皮（Whole Olfactory Epithelium），共获得7份样本：1份来自野生型小鼠，3份来自Amigo1基因敲除纯合子（Amigo1-/-）小鼠，剩余3份来自Amigo1neo/neo纯合子小鼠——该类小鼠的Amigo1基因座中插入了新霉素抗性盒，替代了Amigo1的编码区。