Small hairpin RNA

About: Small hairpin RNA is a research topic. Over the lifetime, 9279 publications have been published within this topic receiving 285471 citations.

Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells

Abstract: Sequence-specific gene silencing by short hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider biomedical use of this approach is hindered by the lack of straightforward methods for achieving uniform expression of shRNAs in mammalian cell cultures. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields virtually homogeneous cell pools containing doxycycline-inducible shRNA elements in a matter of days and with minimal efforts. To ensure immediate utility of this approach for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, HeLa-S3, and U2OS) and mouse (CAD, L929, N2a, NIH 3T3, and P19) cell lines to be compatible with the HILO-RMCE process. Because of its technical simplicity and cost efficiency, the technology will be advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that HILO-RMCE will facilitate a wider range of molecular and cell biology applications by allowing one to rapidly engineer cell populations expressing essentially any transgene of interest.

Soluble CPG15 expressed during early development rescues cortical progenitors from apoptosis.

Abstract: The balance between proliferation and apoptosis is critical for proper development of the nervous system Yet, little is known about molecules that regulate apoptosis of proliferative neurons Here we identify a soluble, secreted form of CPG15 expressed in embryonic rat brain regions undergoing rapid proliferation and apoptosis, and show that it protects cultured cortical neurons from apoptosis by preventing activation of caspase 3 Using a lentivirus-delivered small hairpin RNA, we demonstrate that endogenous CPG15 is essential for the survival of undifferentiated cortical progenitors in vitro and in vivo We further show that CPG15 overexpression in vivo expands the progenitor pool by preventing apoptosis, resulting in an enlarged, indented cortical plate and cellular heterotopias within the ventricular zone, similar to the phenotypes of mutant mice with supernumerary forebrain progenitors CPG15 expressed during mammalian forebrain morphogenesis may help balance neuronal number by countering apoptosis in specific neuroblasts subpopulations, thus influencing final brain size and shape

Fibroblast growth factor receptor 3 inhibition by short hairpin RNAs leads to apoptosis in multiple myeloma.

Abstract: The presence of t(4;14)(p16.3;q32.3) in multiple myeloma cells results in dysregulated expression of the fibroblast growth factor receptor 3 (FGFR3). FGFR3 acts as an oncogene to promote multiple myeloma cell proliferation and antiapoptosis. These encourage the clinical development of FGFR3-specific inhibitors. Three short hairpin RNAs (shRNA) targeting different sites of FGFR3 were selected and subsequently transfected into KMS-11, OPM-2, and NCI-H929 human myeloma cell lines, all of which are characterized by t(4;14) and FGFR3 over expression. The combination of these three shRNAs can effectively inhibit FGFR3 expression in all three cell lines. Sequential immunocytochemistry/fluorescence in situ hybridization was employed to validate that the shRNAs specifically inhibited FGFR3 expression in OPM-2 cells. Decreased expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2) and myeloid cell leukemia sequence 1 (MCL1) proteins and increased staining of Annexin V–positive cells showed that inhibition of FGFR3 induces apoptosis. After confirming down-regulation of FGFR3 by real-time PCR, HU-133 plus 2.0 array was employed to compare the gene expression profile of shRNA-treated sample with that of the control. Besides the down-regulation of FGFR3 , expression of the antiapoptotic genes CFLAR , BCL2 , MCL1 , and some members of NF-κB family decreased, whereas expression of the proapoptotic genes CYC , BID , CASP2 , and CASP6 increased. Microarray results also revealed changes in genes previously implicated in multiple myeloma pathogenesis ( RAS , RAF , IL-6R , and VEGF ), as well as others ( TLR4 , KLF4 , and GADD45A ) not previously linked to multiple myeloma. Our observations indicate that shRNAs can specifically and effectively inhibit FGFR3 expression. This targeted approach may be worth testing in multiple myeloma patients with t(4;14) and FGFR3 overexpression in the future.

RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops.

Abstract: Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops