Biomedical subjects

Tsung-Lin Cheng

Publications and source records attributed to Tsung-Lin Cheng.

5 recordsLinked to original sources

PubMed2006

A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells.

Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.

Animals↗

PubMed2005

A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells.

RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells.

Animals↗

PubMed2005

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference.

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.

Animals↗

PubMed2005

LrrA, a novel leucine-rich repeat protein involved in cytoskeleton remodeling, is required for multicellular morphogenesis in Dictyostelium discoideum.

Cell sorting by differential cell adhesion and movement is a fundamental process in multicellular morphogenesis. We have identified a Dictyostelium discoideum gene encoding a novel protein, LrrA, which composes almost entirely leucine-rich repeats (LRRs) including a putative leucine zipper motif. Transcription of lrrA appeared to be developmentally regulated with robust expression during vegetative growth and early development. lrrA null cells generated by homologous recombination aggregated to form loose mounds, but subsequent morphogenesis was blocked without formation of the apical tip. The cells adhered poorly to a substratum and did not form tight cell-cell agglomerates in suspension; in addition, they were unable to polarize and exhibit chemotactic movement in the submerged aggregation and Dunn chamber chemotaxis assays. Fluorescence-conjugated phalloidin staining revealed that both vegetative and aggregation competent lrrA(-) cells contained numerous F-actin-enriched microspikes around the periphery of cells. Quantitative analysis of the fluorescence-stained F-actin showed that lrrA(-) cells exhibited a dramatically increase in F-actin as compared to the wild-type cells. When developed together with wild-type cells, lrrA(-) cells were unable to move to the apical tip and sorted preferentially to the rear and lower cup regions. These results indicate that LrrA involves in cytoskeleton remodeling, which is needed for normal chemotactic aggregation and efficient cell sorting during multicellular morphogenesis, particularly in the formation of apical tip.

Actins↗

PubMed2005

Simple and efficient DNA vector-based RNAi systems in mammalian cells.

We have constructed four different RNA polymerase III (Pol III)-based expression vectors, containing H1 or U6 promoters from human and mouse, which enable the endogenous production of small RNA transcripts for gene silencing applications. In addition, to facilitate the selection of recombinant clones, we have further improved these vectors by constructing a stuffer of puromycin resistance gene (Puro(r)) between ClaI and HindIII sites, which makes the preparation of vectors easy for rapid and efficient cloning of targeting sequences. A comparative analysis of the silencing efficiency between shRNA, sense-RNA, antisense-RNA, and siRNA showed that both the shRNA and siRNA, but not the sense-RNA and antisense-RNA, dramatically inhibit the targeting gene firefly luciferase activity in mammalian cells. However, there were no significant differences in the inhibition of firefly luciferase expression by shRNA and siRNA expressed from these DNA vectors. In summary, these improved DNA vector-based RNAi systems should provide a simple, convenient, and efficient cloning strategy for studying gene functions in mammalian cells.

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