关键词: RNAi, 大转录本基因, HEK 293, LRP1B

Abstract:

Lipoprotein receptor-related protein 1 B (LRP1B) is one of gigatic receptor subgroup members of lipoprotein receptor family. LRP1B encodes a huge transcript of 16.5 kb. There were difficulties for analysis of LRP1B function due to its huge transcript. In order to study the relation of LRP1B with tumor metastasis, the expression of LRP1B was blocked specially by using RNAi. According to the evaluated standard of the siRNAs and mRNA secondary structure at these target regions, out of the 1 100 candidates, 6 superior ones which locate at different regions of mRNA were selected during designing siRNA sequences. For these 6 targets, shRNA-pSilencer 4.1 recombinant were constructed and transfected into HEK 293 cells. The silencing effects of each siRNA were quantitatively assessed by Semi-Quantitative RT-PCR. It was found that 5 of these 6 shRNA-pSilencer4.1 recombinant could effectively silence the targeted gene LRP1B (>50%). Meanwhile several monoclonal cells, in which the expression of LRP1B was almost completely blocked, had been obtained. The results showed that the method for selecting siRNA, which integrates with mRNA secondary structure, provides a feasible and efficient approach on the design of siRNA of huge gene transcript.