Abstract
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.