One-cell model for inhibiting Cav3.3 mRNA expression by RNA interference

Abstract

To gain insights into the function of Cav3.3 T-type calcium channel in dorsal root ganglion neurons, we designed a one-cell model in dorsal root ganglion neurons that specifically inhibits the expression of Cav3.3 mRNA. We designed three different shRNA sequences and then connected them to pYr-1.1 plasmid and labelled them as pYr-1.1-Cav3.3 shRNA1, pYr-1.1-Cav3.3 shRNA2 and pYr-1.1-Cav3.3 shRNA3, respectively. The correct sequence vector (pYr-1.1-Cav3.3 shRNA3) was then recombined with the pAd/PL-DEST vector into pAd-Cav3.3 shRNA the adenovirus vector. The dorsal root ganglion neurons were infected with the pAd-Cav3.3 shRNA adenovirus vector. After detecting the expression of Cav3.3 mRNA and protein, the pAd-Cav3.3 shRNA adenovirus vector was verified by digestion and sequencing. These results showed that the constructed pAd-Cav3.3 shRNA adenovirus vector could infect dorsal root ganglion cells and inhibit the expression of Cav3.3 mRNA or protein. These results established a one-cell model for further functional study of Cav3.3.

In vitroneurotoxicity by ropivacaine is reduced by silencing Cav3.3 T-type calcium subunits in neonatal rat sensory neurons

“…To investigate the role of Cav3.3 on ropivacaine hydrochloride neurotoxicity, we generated pAd-shRNA and pAd-Cav3.3 adenovirus vector to down-regulate and up-regulate Cav3.3 mRNA expression. According to the previous method [33][34][35], in brief, we designed one shRNA primer according to the Cav3.3 gene of the rats (NM_020084.3) in Genebank: 5 0 -TTTGGCCAGGAAGTTCAACAGTATTCAAGACGTACTGTTGAACTT-CCTGGCTTTTTTG-3 0 ; 5 0 -AGCTCAAAAAAGCCAGGAAGTTCAACA GTACGTCTTGAATACTGTTGAACTTCCTGGC-3 0 (synthesized by Jinsirui Ltd., Nanjing, China). The shRNA primers were annealed to form double strand and connected with pYr-1.1 plasmid (Wuhan Biobuffer Biotech Service Co. Ltd., Wuhan, China) with ligase.…”