Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.

RNA干扰质粒pcDU6的构建及其应用

目的:根据RNAi的原理自主构建一个真核表达的RNAi载体pcDU6。方法:从人的gDNA中克隆出U6promoter(-315～+1),将其与Bgl II和ApaI双酶切的pcDNA3.1(-)质粒大片段酶连,转化JM109大肠杆菌,抽取阳性质粒测序证实,与GFP转染HT1080细胞;干扰肝癌细胞的AFP基因了解干扰效果。结果:pcDU6成功构建转染带有干扰AFP基因表达的RNAi质粒使肝癌细胞的能明显抑制AFP基因的表达(P<0.01),转染质粒后Hep-3B细胞生长明显被抑制(P<0.01)。结论:pcDU6是一个有效的真核表达的RNAi干扰载体。

Regulation of U6 Promoter Activity by Transcriptional Interference in Viral Vector-Based RNAi

The direct negative impact of the transcriptional activity of one component on the second one in c/s is referred to as transcriptional interference （TI）. U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA （shRNA） expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector. Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies. In this research, we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units. We arranged U6 promoter driving .shRNA expression and UbiC promoter in two promoter arrangements. In primary macrophages, we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem. In contrast, PKG promoter had no such negative impact. Instead of enhancing U6 promoter activity, CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter. Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrange- ment-dependent manner.

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.

Establishment of highly efficient transgenic system for black soldier fly(Hermetia illucens)

The black soldier fly(BSF),Hermetia illucens,is a promising insect for miti-gating solid waste problems as its larvae are able to bioconvert organic waste into valuable biomass.We recently reported a high-quality genome assembly of the BSF;analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of bioconversion.To enable genetic manip-ulation of the BSF,we have established the first transgenic methods for this economically important insect.We cloned and identified the ubiquitous actin5C promoter(Hiactin5C-p3k)and 3 endogenous U6 promoters(HiU6:1,HiU6:2,and HiU6:3).The Hiactin5C pro-moter was used to drive expression of a hyperactive variant of the piggyBac transposase,which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type transposase.Furthermore,we evaluated the 3 HiU6 promoters using this trans-genic system.HiU6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)development.Overall,our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.