Suppression of prion protein in livestock by RNA interference

Given the difficulty of applying gene knockout technology to species other than mice, we decided to explore the utility of RNA interference (RNAi) in silencing the expression of genes in livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability to suppress the expression of caprine and bovine prion protein (PrP). Lentiviral vectors were used to deliver a transgene expressing GFP and an shRNA targeting PrP into goat fibroblasts. These cells were then used for nuclear transplantation to produce a cloned goat fetus, which was surgically recovered at 81 days of gestation and compared with an age-matched control derived by natural mating. All tissues examined in the cloned fetus expressed GFP, and PCR analysis confirmed the presence of the transgene encoding the PrP shRNA. Most relevant, Western blot analysis performed on brain tissues comparing the transgenic fetus with control demonstrated a significant (>90%) decrease in PrP expression levels. To confirm that similar methodologies could be applied to the bovine, recombinant virus was injected into the perivitelline space of bovine ova. After in vitro fertilization and culture, 76% of the blastocysts exhibited GFP expression, indicative that they expressed shRNAs targeting PrP. Our results provide strong evidence that the approach described here will be useful in producing transgenic livestock conferring potential disease resistance and provide an effective strategy for suppressing gene expression in a variety of large-animal models

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Alternative initiation and splicing in dicer gene expression in human breast cells

INTRODUCTION: Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene. METHOD: We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo. RESULTS: We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein. CONCLUSION: Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells

Identification and Validation of ERK5 as a DNA Damage Modulating Drug Target in Glioblastoma

YesBrain tumours kill more children and adults under 40 than any other cancer, with approximately half of primary brain tumours being diagnosed as high-grade malignancies known as glioblastomas. Despite de-bulking surgery combined with chemo-/radiotherapy regimens, the mean survival for these patients is only around 15 months, with less than 10% surviving over 5 years. This dismal prognosis highlights the urgent need to develop novel agents to improve the treatment of these tumours. To address this need, we carried out a human kinome siRNA screen to identify potential drug targets that augment the effectiveness of temozolomide (TMZ)-the standard-of-care chemotherapeutic agent used to treat glioblastoma. From this we identified ERK5/MAPK7, which we subsequently validated using a range of siRNA and small molecule inhibitors within a panel of glioma cells. Mechanistically, we find that ERK5 promotes efficient repair of TMZ-induced DNA lesions to confer cell survival and clonogenic capacity. Finally, using several glioblastoma patient cohorts we provide target validation data for ERK5 as a novel drug target, revealing that heightened ERK5 expression at both the mRNA and protein level is associated with increased tumour grade and poorer patient survival. Collectively, these findings provide a foundation to develop clinically effective ERK5 targeting strategies in glioblastomas and establish much-needed enhancement of the therapeutic repertoire used to treat this currently incurable disease

Identification and validation of ERK5 as a DNA damage modulating drug target in glioblastoma

Brain tumours kill more children and adults under 40 than any other cancer, with approximately half of primary brain tumours being diagnosed as high-grade malignancies known as glioblastomas. Despite de-bulking surgery combined with chemo-/radiotherapy regimens, the mean survival for these patients is only around 15 months, with less than 10% surviving over 5 years. This dismal prognosis highlights the urgent need to develop novel agents to improve the treatment of these tumours. To address this need, we carried out a human kinome siRNA screen to identify potential drug targets that augment the effectiveness of temozolomide (TMZ)—the standard-of-care chemotherapeutic agent used to treat glioblastoma. From this we identified ERK5/MAPK7, which we subsequently validated using a range of siRNA and small molecule inhibitors within a panel of glioma cells. Mechanistically, we find that ERK5 promotes efficient repair of TMZ-induced DNA lesions to confer cell survival and clonogenic capacity. Finally, using several glioblastoma patient cohorts we provide target validation data for ERK5 as a novel drug target, revealing that heightened ERK5 expression at both the mRNA and protein level is associated with increased tumour grade and poorer patient survival. Collectively, these findings provide a foundation to develop clinically effective ERK5 targeting strategies in glioblastomas and establish much-needed enhancement of the therapeutic repertoire used to treat this currently incurable disease

Stilbene derivatives promote Ago2-dependent tumour-suppressive microRNA activity

It is well known that natural products are a rich source of compounds for applications in medicine, pharmacy, and biology. However, the exact molecular mechanisms of natural agents in human health have not been clearly defined. Here, we demonstrate for the first time that the polyphenolic phytoalexin resveratrol promotes expression and activity of Argonaute2 (Ago2), a central RNA interference (RNAi) component, which thereby inhibits breast cancer stem-like cell characteristics by increasing the expression of a number of tumour-suppressive miRNAs, including miR-16, -141, -143, and -200c. Most importantly, resveratrol-induced Ago2 resulted in a long-term gene silencing response. We also found that pterostilbene, which is a natural dimethylated resveratrol analogue, is capable of mediating Ago2-dependent anti-cancer activity in a manner mechanistically similar to that of resveratrol. These findings suggest that the dietary intake of natural products contributes to the prevention and treatment of diseases by regulating the RNAi pathway

Inducible Transgenic Rat Model for Diabetes Mellitus Based on shRNA-Mediated Gene Knockdown

The rat is an important animal model in biomedical research, but gene targeting technology is not established for this species. Therefore, we aimed to produce transgenic knockdown rats using shRNA technology and pronuclear microinjection. To this purpose, we employed a tetracycline-inducible shRNA expression system targeting the insulin receptor (IR). Doxycycline (DOX) treatment of the resulting transgenic rats led to a dose-dependent and reversible increase in blood glucose caused by ubiquitous inhibition of IR expression and signalling. We could neither detect an interferon response nor disturbances in microRNA processing after DOX treatment excluding toxic effects of shRNA expression. Low dose DOX treatment induced a chronic state of diabetes mellitus. In conclusion, we have developed a technology which allows the specific, inducible, and reversible suppression of any gene of interest in the rat. Our first transgenic rat line generated with this method represents an inducible model for diabetes mellitus

Planting the Seeds of a New Paradigm

RNA-mediated gene silencing has emerged in recent years as an important mechanism for regulating gene expression. Some of the key discoveries have been made in plant