This is a summary in recent progress of small interference technology.
1: Curr Protoc Mol Biol. 2008 Oct;Chapter 3:Unit3.13.
Ribonucleases.
Nichols NM, Yue D.
Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed. (c) 2008 by John Wiley & Sons, Inc.
2: J Dig Dis. 2008 Nov;9(4):228-37.
Suppression of growth of pancreatic cancer cell and expression of vascular endothelial growth factor by gene silencing with RNA interference.
Wang J, Shi YQ, Yi J, Ye S, Wang LM, Xu YP, He M, Kong XM.
Division of General Surgery, Renji Hospital, Shanghai, China.
OBJECTIVE: To explore the anti-angiogenesis and tumor cell growth suppressive effects resulted from gene silencing by RNAi in BxPC-3 human pancreatic cancer cells. METHODS: The designation and transfection of vascular endothelial growth factor (VEGF)-siRNA lentivirus was carried out in vitro. Real-time PCR and western blot were conducted to measure the expression levels of VEGF mRNA and protein. Flow cytometry was employed to evaluate cell apoptosis and cell death. A lactate dehydrogenase (LDH) assay was used to assess the cytotoxicity of VEGF-siRNA. A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to picture the cellular growth. For the in vivo study, BxPC-3 cells were injected subcutaneously into nude mice to form xenografts. The mice were divided into three groups according to the intervention used. The control group, the negative control group and the knockdown group of mice were injected with saline, an empty lentivirus vehicle and lentivirus carrying VEGF-siRNA, respectively. None of the mice died during the study. When these mice were killed, the xenografts were collected and the tumor sizes of the different groups were compared. Finally, immunohistochemistry was used to assess the VEGF expression level and microvascular density. RESULTS: After the transfection of VEGF-siRNA lentivirus, the cellular expression of VEGF mRNA decreased to 50% of the control and the VEGF protein in the BxPC-3 cells decreased to 30% of the control. Apoptosis and cell death increased after transfection of the VEGF-siRNA lentivirus. The LDH assay showed high cytotoxicity induced by VEGF-siRNA lentivirus transfection. The MTT assay showed slower cellular growth in the knockdown cells. Tumor growth suppression was observed in nude mice that had received the VEGF-siRNA lentivirus transfection, and the tumor sizes of the xenografts in this group were clearly smaller than those in other two groups. VEGF expression and microvascular density were significantly decreased. CONCLUSION: Vascular endothelial growth factor gene silencing via VEGF-siRNA can effectively inhibit the production of VEGF and exert an anti-angiogenesis and tumor cell growth suppressive effect both in vitro and in vivo.
3: Int J Dev Biol. 2008;52(7):913-23.
Maternal RNAs encoding transcription factors for germline-specific gene expression in Drosophila embryos.
Yatsu J, Hayashi M, Mukai M, Arita K, Shigenobu S, Kobayashi S.
Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki, Japan.
In early Drosophila embryos, germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm contains the maternal factors required for germline development. It has been proposed that germline-specific gene expression is initiated by the function of maternal factors that are enriched in the germ plasm. However, such factors have remained elusive. Here, we describe a genome-wide survey of maternal transcripts that encode for transcription factors and are enriched in the germ plasm. We isolated pole cells from blastodermal embryos by fluorescence-activated cell sorting (FACS) and then used these isolated cells in a microarray analysis. Among the 835 genes in the Gene Ontology (GO) category transcription regulator activity listed in FlyBase, 68 were found to be predominantly expressed in pole cells as compared to whole embryos. As the early pole cells are known to be transcriptionally quiescent, the listed transcripts are predicted to be maternal in origin. Our in situ hybridization analysis revealed that 27 of the 68 transcripts were enriched in the germ plasm. Among the 27 transcripts, six were found to be required for germline-specific gene expression of vasa and/or nanos by knockdown experiments using RNA interference (RNAi). The identified transcripts encode a transcriptional activator (ovo), components of the transcriptional initiation complexes (Trf2, bip2 and Tif-IA), and the Ccr4-Not complex (CG31716 and l(2)NC136). Our study demonstrates that germ plasm contains maternal transcripts encoding transcriptional regulators for germline-specific gene expression in pole cells.