Section on Molecular Neurobiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Int J Mol Sci. 2022 Aug 21;23(16):9462. doi: 10.3390/ijms23169462.
A popular approach to spatiotemporally target genes using the /Cre recombination system is stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) in specific neuronal structures. Here, we report that AAV_Cre microinjection in the ventral tegmental area (VTA) of ErbB4 Cyt-1-floxed (ErbB4 Cyt-1) mice at titers commonly used in the literature (~10-10 GC/mL) can have neurotoxic effects on dopaminergic neurons and elicit behavioral abnormalities. However, these effects of AAV_Cre microinjection are independent of ErbB4 Cyt-1 recombination because they are also observed in microinjected wild-type (WT) controls. Mice microinjected with AAV_Cre (10-10 GC/mL) exhibit reductions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) expression, loss of dopaminergic neurons, and they behaviorally become hyperactive, fail to habituate in the open field and exhibit sensorimotor gating deficits compared to controls microinjected with AAV_GFP. Importantly, these AAV_Cre non-specific effects are: (1) independent of serotype, (2) occur with vectors expressing either Cre or Cre-GFP fusion protein and (3) preventable by reducing viral titers by 1000-fold (10 GC/mL), which retains sufficient recombination activity to target floxed genes. Our studies emphasize the importance of including AAV_Cre-injected WT controls in experiments because recombination-independent effects on gene expression, neurotoxicity and behaviors could be erroneously attributed to consequences of gene ablation.
一种使用 Cre 重组系统对基因进行时空靶向的常用方法是立体定位微注射表达 Cre 重组酶的腺相关病毒（AAV）（AAV_Cre）到特定神经元结构中。在这里，我们报告在 ErbB4 Cyt-1-floxed（ErbB4 Cyt-1）小鼠腹侧被盖区（VTA）中以文献中常用的滴度（~10-10GC/mL）进行 AAV_Cre 微注射会对多巴胺能神经元产生神经毒性作用并引发行为异常。然而，这些 AAV_Cre 微注射的影响独立于 ErbB4 Cyt-1 重组，因为在微注射的野生型（WT）对照中也观察到了这些影响。用 AAV_Cre（10-10GC/mL）微注射的小鼠表现出酪氨酸羟化酶（TH）和多巴胺转运蛋白（DAT）表达减少、多巴胺能神经元丧失，并且行为变得过度活跃，在旷场中无法习惯化，并表现出感觉运动门控缺陷，与微注射 AAV_GFP 的对照相比。重要的是，这些 AAV_Cre 的非特异性作用是：（1）独立于血清型，（2）与表达 Cre 或 Cre-GFP 融合蛋白的载体有关，（3）通过将病毒滴度降低 1000 倍（10GC/mL）可预防，这保留了足够的重组活性以靶向 floxed 基因。我们的研究强调了在实验中包括 AAV_Cre 微注射 WT 对照的重要性，因为基因表达、神经毒性和行为的与重组无关的影响可能会被错误地归因于基因缺失的后果。
