BLT Stages

Abstract Bachelor Project FBT 2018-2019: Optimisation of the knockdown-efficiency based on shRNA

RNA interference (RNAi) is a mechanism in which small non-coding RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA. mRNA is often silenced using short hairpin RNA (shRNA) technology. Although shRNA provides a useful tool for gene repression, an efficient knockdown of two transcription factors important in human NK cell maturation is still not achieved. Therefore, screening of these transcription factors for new shRNA target sequences was conducted, whereby 11 shRNA target sequences were selected. Besides, shRNA was imbedded into endogenous microRNA contexts in order to improve knockdown efficiency.

First, shRNA target sequences were transformed into a miR-E optimized backbone (miR-E). Then, shRNA and miR-E were introduced in the pLKO.1-eGFP and pINDUCER11-RUG, respectively, by standard molecular cloning. For optimization, the miR-E also needed to be cloned in the pINDUCER-RMG. These vectors were then transfected into virus-producing HEK- 293T cells, after which the viral particles were used to transduce the NKL cell line, whereby transduction efficiency as well as knockdown efficiency was determined. All of the constructs, except for two, are successfully cloned in the respective vectors and pINDUCER11-miR-RUG, pINDUCER-miR-RMG and pLKO.1-shRNA-eGFP viruses were made. In the NKL cell line, transduction with the pINDUCER11-miR-RUG and pINDUCER-miR-RMG viruses was not successful. Therefore differences in knockdown efficiency and stability could not be determined between the two types of constructs. Due to the time frame of this project, knockdown efficiency of the new pLKO.1-shRNA-eGFP viruses could not be determined. Further research is required to test the knockdown efficiency and stability of the shRNAs on NKL cells. Furthermore, miR-E expression vectors and transduction systems need further optimization.