Data_Sheet_1_The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform.docx

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1–12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13–16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1–12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

编码突触后支架蛋白的SH3与多个锚蛋白重复结构域3（SH3 and multiple ankyrin repeat domains 3, SHANK3）的基因变异与脑部疾病相关。目前部分已构建的Shank3基因敲除（knock-out, KO）品系的靶向等位基因中，携带有新霉素抗性（Neo）筛选盒，该元件可能会干扰邻近基因的正常表达，但此前尚未对此展开深入研究。本团队此前曾报道，在通过用Neo筛选盒替换Shank3基因第13~16号外显子构建的Shank3B KO小鼠脑组织中，Shank3基因第1~12号外显子的mRNA表达水平出现了异常升高。本研究中，我们证实Shank3B KO小鼠脑组织中升高的Shank3 mRNA会翻译产生一种分子量约为60 kDa的异常SHANK3蛋白亚型（Shank3-N），该亚型无法正常定位至突触区室。功能层面而言，过表达Shank3-N会改变培养神经元的树突棘形态。尤为关键的是，Shank3B KO小鼠体内的Shank3-N表达并非对全长SHANK3蛋白表达下调的代偿性反应：在对全长SHANK3的表达水平进行标准化校正后，我们仍可在脑组织中检测到Shank3-N的表达。此外，另一株Shank3 KO品系（Shank3 gKO）的Shank3基因外显子缺失位点与Shank3B KO小鼠相似，但未携带Neo筛选盒；该品系小鼠脑组织中Shank3基因第1~12号外显子的mRNA表达水平低于野生型小鼠，且未检测到Shank3-N的存在。除此之外，Shank3B KO小鼠纹状体中15号染色体上Shank3基因邻近区域的基因表达水平发生了改变，而Shank3 gKO小鼠则未出现该现象。上述研究结果表明，Neo筛选盒在Shank3B KO小鼠中存在潜在的脱靶效应。