Short hairpin RNA (shRNA)-mediated gene regulation is a commonly used technique for gene manipulation. An efficient and safe delivery system is indispensable when shRNA is delivered into living organisms for gene therapy. Previous studies have proved that DNA and protein can be delivered into dendritic cells (DCs) by non-pathogenic Saccharomyces cerevisiae without being degraded. CD40 is closely related to apoptosis of tumor cells and some immune mechanisms. In this study, we demonstrated that recombinant yeast S. cerevisiae efficiently delivered the shRNA of immune-associated gene (CD40) into mouse intestinal DCs via oral administration. Western blot analysis of isolated intestinal DCs indicated that the inhibition of CD40 gene expression reached up to 56–91%. The secretion of cytokines such as interleukin-2 (IL-2), IL-6, IL-10, IL-12, tumor necrosis factor-α and interferon-γ in intestinal DCs had varying degrees of changes. In conclusion, we found that orally administered recombinant yeast can be used as an efficient shRNA delivery system for intestinal DC-specific gene silencing and immunomodulation in vivo.

1 CRISPR/Cas9 system has become a new versatile technology for genome engineering. It 2 utilizes a single guide RNA (sgRNA) to recognize target sequences in genome function, and 3 activates Cas9 endonucleases to cut the locus. In this study, we designed two target sites from 4 conserved regions of vitamin D receptor (VDR) gene in mammalian cells, which cover more 5 than 17 kb of chromosome region depending on the species. The efficacy of single sgRNA 6 mediated gene specific modification was about 22% to 36%. Concurrently, targeted deletions 7 of the intervening genomic segments were generated in chromosomes when the two sgRNAs 8 worked simultaneously. The large genomic DNA segments ranging from 17.8Kb to 23.4 Kb 9 could be precisely deleted in human and mouse chromosomes. Furthermore, the expression 10 level of 24-hydroxylase (CYP24A1) regulated by VDR was significantly increased in cells 11 treated with VDR CRISPR/Cas9 vectors. This study showed that CRISPR/Cas9 system can be 12 employed to generate large genomic segment deletions in different species, providing 13 sgRNAs are designed within conserved regions. 14

ABSTRACT. Herp, a mammalian protein with a ubiquitin-like domain, can be strongly upregulated by endoplasmic reticulum (ER) stress during ERassociated protein degradation. However, the other cellular functions of Herp remain unclear. We explored the effect of Herp on ER stress and inflammatory responses in RAW 264.7 macrophages that had been exposed to tunicamycin or thapsigargin. We successfully constructed recombinant lentiviral vectors for Herp short-hairpin RNA (shRNA) expression to better understand the contribution made by Herp to other signaling pathways. Western blotting revealed that the recombinant Herp lentiviral shRNA vector significantly inhibited the expression of the Herp protein in the thapsigargintreated RAW 264.7 macrophages. The reverse transcription quantitative polymerase chain reaction results showed that knockdown Herp inhibited the expression of ER stress-related genes during exposure to tunicamycin or thapsigargin. In RAW 264.7 macrophages, knockdown Herp markedly attenuated the expression of inflammatory cytokines when exposed to tunicamycin; however, it strongly enhanced the expression of inflammatory cytokines when exposed to thapsigargin. We concluded that Herp lentiviral shRNA vectors had been successfully constructed; knockdown Herp inhibited ER stress and had a different effect on inflammatory responses in RAW 264.7 macrophages depending on whether they were exposed to tunicamycin or thapsigargin.

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