BacMam 是以桿狀病毒為基礎所發展的哺乳類細胞基因遞送載體。我們首先嘗試以 BacMam 作為神經細胞的基因遞送載體，發現 BacMam 雖然對於海馬迴神經元的轉導效率低，但對未分化的神經前驅細胞反而可以進行有效率的基因轉導：於 BacMam 轉導神經前驅細胞後以 5 mM 的組蛋白去乙醯酶抑制劑 NaBt 進行處理可提高轉導效率至 80%。除此之外，我們亦發現 alpha-synuclein (αSyn) 和綠螢光蛋白融合 (αSyn-EGFP) 後能夠提高綠螢光蛋白基因的表達量以及延長外源蛋白的表現時間。因此，帶有 αSyn-EGFP 基因的 BacMam 可做為神經細胞分化或標定之工具。於是我們接著使用分別帶有 αSyn-EGFP 與 neuroligin-1-EGFP (NLG1-EGFP) 的 BacMam 進行轉導神經前驅細胞，在神經分化過程中追蹤 αSyn 或 NLG1 的分布情形。結果顯示過度表達的 αSyn-EGFP 揭示了能夠長期表達的能力，而 NLG1-EGFP 則無，並且顯示 αSyn 位於細胞質和細胞核中，而 NLG1 則是均勻分布於細胞膜上但並非集中於後突觸膜上。這是第一次使用 BacMam 將基因遞送入神經前驅細胞衍生的神經細胞中，並且成功在體外神經前驅細胞發育過程中追蹤 αSyn 位置。於目前的文獻中因 NLG1 的派送訊息還尚未清楚，於是我們構築了不同區域的 NLG1，包含全長、N 端剃除和 C 端剃除等融合螢光蛋白基因的 BacMam 載體。我們的結果推測：可能的派送訊息區域位於 NLG1 之 C 端 772-804 胺基酸。除此之外，在 vAcCMV-NLG1 (1-843)-Lir-EGFP 轉導的細胞當中，我們發現 NLG1 上可能存在一尚未發現的選擇性剪接異體。我們經由分析綠螢光蛋白分佈、西方墨點法、選擇性剪接預測軟體、反轉錄聚合酶鏈式反應和序列比對等方法證明此選擇性剪接位的存在。結果顯示 NLG1 (1-843)-Lir-EGFP mRNA 會經由選擇性剪接作用導致 Lir-IRES 的剃除，產生 NLG1 (1-763)-EGFP 融合產物，於 NLG1 上少了 81 個胺基酸且與西方墨點法所臆測的分子量一致。更進一步，我們發現一種新型的 NLG1 內生性選擇性剪接異體，轉譯出 C 端剃除 17 個胺基酸的 NLG1 異體。這是首度發現 NLG1 之 C 端存有內生性的選擇性剪接位，對其生理功能則有待進一步確認。

BacMam is a baculovirus-based gene delivery vector for mammalian cells. In the beginning, we used BacMam as a delivery tool for transferring genes into neurons. We found out that the transduction efficiency of BacMam is low in hippocampal neurons, while it can efficiently transduce undifferentiated neural stem/progenitor cells (rNSPCs) in vitro. After treatment with 5mM NaBt, an inhibitor of histone deacetylases (HDACi), the transduction efficiency of BacMam in rNSPCs can be enhanced up to 80%. In addition, the expression of EGFP can be elevated and the duration of foreign protein expression can be prolonged by fused with alpha-synuclein (formed αSyn-EGFP). Thus, αSyn-EGFP BacMam has the potential to become a tool for neuronal differentiation or labelling. We then transduced rNSPCs with αSyn-EGFP and neuroligin-1-EGFP (NLG1-EGFP) BacMam, respectively, and tracked the transport of αSyn or NLG1 during neuronal differentiation. The results showed that overexpression of αSyn-EGFP but not NLG1-EGFP revealed the property of long-term expression, and indicated that αSyn was located in both the cytoplasm and the nucleus, whereas NLG1 was stably localized to the plasma membrane (not cluster in the postsynaptic membrane). This is the first time using BacMam to deliver genes into rNSPC-derived neurons and successfully track αSyn during rNSPCs development in vitro. Because of the sorting signal of NLG1 is not fully understood in the current literature. We constructed different region of NLG1 fused with fluorescence gene, including full-length, N-terminal truncated and C-terminal truncated into BacMam vector. According to our data, we can speculate that the dendritic sorting signal located within amino acid residues 772–804 of NLG1 C-terminus. Moreover, we identified that NLG1 may contain a previously undiscovered alternative splicing variant in vAcCMV-NLG1 (1-843) -Lir-EGFP transduced cells. Using EGFP distribution, Western blotting, alternative splice site predictor, RT-PCR, and sequence alignment, we demonstrated the presence of alternative splicing site. The results indicated that NLG1 (1-843)-Lir-EGFP mRNA undergoes alternative splicing, resulting in the Lir-IRES skipping and producing NLG1 (1-763)-EGFP fusion transcript in which 81 amino acid residues deleted from the C terminal of NLG1 and consistent with the estimated molecular weight determined by Western blotting. Furthermore, we identified a novel endogenous alternative splice variant of NLG1 which 17 amino acid residues deleted from the C terminus. This is the first study to identify that endogenous NLG1 has alternative splice sites at C terminus, the physiological function of this variant remains to be clarified.

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