Short Hairpin RNA Expression Research Articles

An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.

Depletion of L3MBTL1, a PcG Gene Deleted in Patients with 20q-Associated with Hematologic Malignancies, Accelerates Erythroid Differentiation of Hematopoietic Progenitor Cells

L3MBTL1 is a polycomb gene located in 20q12, within the common deleted region identified in patients with 20q deletion associated with polycythaemia vera (PV), myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). L3MBTL1 is expressed within CD34+ haematopoietic progenitor cells from which myeloid malignancies arise and its Drosophila homologue encodes a tumour suppressor protein. L3MBTL1 represents then a candidate target gene in 20q deletion patients. To gain insight into the role of L3MBTL1 in hematopoiesis we knocked down the level of L3MBTL1 mRNA through lentiviral expression of short hairpin RNAs in CD34+ hematopoietic stem/progenitor cells isolated from human cord blood (CB) cells. We achieved an approximately 80% drop in endogenous L3MBTL1 mRNA level, as determined using quantitative RT-PCR. Transduced and sorted CD34+ GFP+ CB cells were plated in liquid cultures and induced to cytokine-driven differentiation. The effect of L3MBTL1-knock down was assessed by colony assays and by fluorescence-activated cell sorting (FACS), using lineage–specific cell surface markers. We demonstrate that the knock down (KD) of L3MBTL1 remarkably accelerates the differentiation of hematopoietic CD34+ cells into erythrocytes. The relative percentage of mature erythroid precursors cells, defined as CD71+ and Glycophorin A+ cells, consistently increased in the L3MBTL1-KD population, as the CD34+ cells reached this advanced stage of erythroid differentiation in fewer days than control cells. As confirmation, Giemsa staining after cytospin preparations of L3MBTL1-KD cells showed more mature morphology compared to the control cells, and benzidine staining revealed many more Hb containing-positive cells in the L3MBTL1-depleted cell population compared to the control. Monitoring of globin gene expression demonstrated that L3MBT-Knock down is involved in the regulation of only a subset of these genes, primarily the expression of the epsilon and zeta globin genes, the embryonic globin genes belonging to the beta and alpha globin gene clusters respectively. In addition to these effects a significant slowing of proliferation was seen, which likely reflects the increased differentiation of these cells. We have previously identified a role for L3MBT in binding histones H1 and H4 that contain monomethylated and dimethylated lysine residues H1k2b and H4k20 (Kalakonda et al. 2008) and compacting chromatin (Trojer et al. Cell 2007). Precisely how this feature of L3MBTL1 function in regulating the erythroid differentiation of hematopoietic cells is unknown. We however link lack of L3MBTL1 with the possible pathogenesis of PV associated with 20q deletion. We will present additional data that attempt to define the kinetics of alpha-like and beta-like globin gene expression during the erythroid maturation of L3MBTL1-KD cells in culture and to investigate a supposed synergism with the known molecular pathways of normal and malignant erythroid differentiation.

Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn 2+ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg 2+ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.

Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression.

RNA interference (RNAi) is an effective tool for studying gene function in oocytes, but no studies have targeted somatic cells of primary cultured cumulus cell-oocyte complexes (COCs). This is probably due to difficulty in introducing RNAi-inducing molecules, such as a short-hairpin RNA (shRNA) gene, into COCs by commonly used transfection reagents. We therefore tested whether a developmental process of intact COCs could be suppressed by adenovirus-mediated shRNA expression. Has2, encoding hyaluronan synthase 2, was selected as the target transcript, because the process of cumulus expansion depends upon expression of Has2 mRNA and this process is easily evaluated in vitro. Intact COCs were infected with replication-incompetent adenoviruses containing an expression sequence of shRNA targeting either Has2 (Has2 shRNA) or a control transcript not expressed in cumulus cells, and the effects on epidermal growth factor (EGF)-stimulated cumulus expansion were determined. Has2 shRNA expression suppressed Has2 mRNA levels in COCs by more than 70%, without affecting expression levels of Ptgs2, Ptx3, Tnfaip6 mRNAs, which are also required for cumulus expansion, or other transcripts not related to expansion. Interestingly, levels of Areg and Ereg mRNAs were decreased in COCs expressing Has2 shRNA when compared with those in controls, while Btc mRNA levels remained unaffected. Furthermore, the degree of cumulus expansion by Has2 shRNA-expressing COCs was significantly less than that of controls. Thus adenovirus-mediated introduction of shRNA produces specific gene silencing and a phenotype in intact COCs, providing proof of principle that this method will be a helpful tool for understanding mechanisms of COC development.

Targeted and Conditional Gene Expression Workshop, 8th International Conference on Zebrafish Development and Genetics

Several technologies for studying gene function in zebrafish embryos, larvae, and adults were discussed at the Targeted and Conditional Gene Expression workshop. The workshop included six oral presentations followed by a community discussion of current approaches, their limitations, and possible solutions. The research talks focused on new methods for controlling the activity of endogenous or exogenous genes through genetic and/or chemical strategies. Approaches for conditionally regulating endogenous gene function included short-hairpin RNA (shRNA) expression, reversible transposon-induced mutations, and caged morpholinos. Technologies for controlling exogenous gene activity included a chemically inducible progesterone receptor-based transactivator, the Cre-LoxP system, and estrogen receptor fusion proteins. General considerations for using the Gal4/UAS system in zebrafish were also discussed.

Modulating endogenous gene expression of mammalian cells via RNA–small molecule interaction

RNA interference (RNAi) has emerged as a powerful technology to silence arbitrary genes by designing small RNA constructs based on the targeted messenger RNA sequences. We recently developed a small molecule-controlled RNAi gene switch that combined the molecular recognition by in vitro selected RNA aptamers with versatile gene silencing by small interfering RNAs, and demonstrated for the first time, posttranscriptional modulation of RNAi through direct RNA–small molecule interaction. In this report, we describe the first application of this technology to regulate an endogenous gene in mammalian cells. As a proof-of-concept demonstration we chose to modulate expression of albumin—serum protein produced by the liver. We designed and constructed a theophylline aptamer-fused short hairpin RNA (shRNA) expression vector targeting albumin mRNA in hepatic (HepG2) cells. Transfection of HepG2 cells with the aptamer–shRNA expression vector allowed to control albumin gene expression by adding theophylline into the culture media in dose dependent fashion.

Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction

We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P(1)R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P(2)R antagonist JTE-013. The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P(1)R and S1P(2)R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P(1)R. However, an S1P(1)R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P(2)R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P(2)R function further enhances the migration of NPCs toward a brain infarction.

Accumulation of hypoxia-inducible factor-1 protein and its role in the differentiation of myeloid leukemic cells induced by all-trans retinoic acid

The clinical activities of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia, have triggered extensive studies aimed at defining the mechanisms by which this compound induces differentiation of leukemic cells. Recent studies show that hypoxia-inducible factor-1 alpha (HIF-1 alpha) contributes to the differentiation of acute myeloid leukemia cells via transcriptional activity-independent mechanisms. We investigated whether all-trans retinoic acid affects HIF-1 alpha protein and whether this has a role in all-trans retinoic acid-induced differentiation. The acute myeloid leukemia cell lines NB4 and U937 were treated with all-trans retinoic acid, and HIF-1 alpha/HIF-1 beta mRNA and proteins were measured respectively by real-time quantitative reverse transcriptase polymerase chain reaction and western blotting. To investigate the role of HIF-1 alpha in all-trans retinoic acid-induced differentiation, NB4 cells, U937 cells, U937 cells in which HIF-1 alpha was induced by the withdrawal of tetracycline and U937 cells with stable expression of specific short hairpin RNA against HIF-1 alpha, Runx1, C/EBP alpha and PU.1, were treated with all-trans retinoic acid and/or the hypoxiamimetic agent cobalt chloride (CoCl(2)). Cellular differentiation was evaluated by morphological criteria and myeloid differentiation antigens. all-trans retinoic acid rapidly increased endogenous and inducible expressed or CoCl(2)-stabilized HIF-1 alpha protein in leukemic cells under normoxia. Importantly, suppression of HIF-1 alpha expression by specific short hairpin RNA partially but significantly inhibited all-trans retinoic acid-induced differentiation of the U937 cell line. Reciprocally, the differentiation induced by all-trans retinoic acid was significantly enhanced by conditional HIF-1 alpha induction and HIF-1 alpha-stabilizing CoCl(2) treatment. Furthermore, knock-down of PU.1, Runx1 and C/EBP alpha, three transcriptional factors crucial for normal hematopoiesis, greatly inhibited the differentiation cooperation of all-trans retinoic acid and HIF-1 alpha induction. This work provides the first demonstration that HIF-1 alpha, a protein rapidly responsive to all-trans retinoic acid, plays a role in all-trans retinoic acid-induced differentiation of leukemic cells. These observations shed new light on the molecular mechanisms underlying all-trans retinoic acid-induced differentiation of acute myeloid leukemia cells.

Spermatogonial Stem Cell Self-Renewal Requires OCT4, a Factor Downregulated During Retinoic Acid-Induced Differentiation

The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. Octamer-4 (OCT4) plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs. Here, we show that OCT4 is required for SSC maintenance in culture and for colonization activity following cell transplantation, using lentiviral-mediated short hairpin RNA expression to knock down OCT4 in an in vitro model for SSCs ("germline stem" [GS] cells). Expression of promyelocytic leukemia zinc-finger (PLZF), a factor known to be required for SSC self-renewal, was not affected by OCT4 knockdown, suggesting that OCT4 does not function upstream of PLZF. In addition to developing a method to test specific gene function in GS cells, we demonstrate that retinoic acid (RA) triggers GS cells to shift to a differentiated, premeiotic state lacking OCT4 and PLZF expression and colonization activity. Our data support a model in which OCT4 and PLZF maintain SSCs in an undifferentiated state and RA triggers spermatogonial differentiation through the direct or indirect downregulation of OCT4 and PLZF. The current study has important implications for the future use of GS cells as an in vitro model for spermatogonial stem cell biology or as a source of embryonic stem-like cells. Disclosure of potential conflicts of interest is found at the end of this article.

Prevention of interferon-stimulated gene expression using microRNA-designed hairpins

RNA interference allows selective gene silencing, and is widely used for functional analysis of individual genes in vertebrate cells and represents an attractive therapeutic option for treating central nervous system diseases. However, growing evidence exists that the expression of short hairpin RNAs (shRNAs) can trigger cellular immune response resulting in unspecific cellular phenotypes and severe side effects. We found that lentiviral vector (LV)-mediated expression of shRNAs in primary cortical cultures resulted in strong expression of the interferon-stimulated gene oligoadenylate synthetase 1 (Oas1), which was accompanied by accelerated apoptosis and substantial net neuron loss. Modification of the shRNA construct by implementing features of the naturally occurring microRNA-30 (miR-30) precursor avoided Oas1 induction in transduced primary cultures, whereby modification of the passenger strand seems to be a crucial feature to circumvent interferon-stimulated gene expression. This work represents the first experimental study showing that an miR-30-based shRNA construct prevents Oas1 pathway associated off-target effects, which we consider as an essential prerequisite for shRNA use in future gene therapeutic approaches.

Suppression of hepatitis C virus replication by baculovirus vector-mediated short-hairpin RNA expression

Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. Here, we report on the inhibition, by baculovirus-based vector-derived shRNAs, of core-protein expression in full-length hepatitis C virus (HCV) replicon cells. shRNAs were designed to target the highly conserved core region of the HCV genome. In particular, the core-shRNA452 containing nucleotides 452–472, as the target in the HCV core gene, dramatically inhibited the expression of the HCV core protein in replicon cells. Furthermore, HCV core-protein expression was inhibited more strongly by the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped baculovirus vector than by the wild-type baculovirus vector.

Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.

Inhibition of Mouse GPM6A Expression Leads to Decreased Differentiation of Neurons Derived from Mouse Embryonic Stem Cells

Glycoprotein M6A (GPM6A) is known as a transmembrane protein and an abundant cell surface protein on neurons in the central nervous system (CNS). However, the function of GPM6A is still unknown in the differentiation of neurons derived from embryonic stem (ES) cells. To investigate the function of GPM6A, we generated knockdown mouse ES cell lines (D3m-shM6A) using a short hairpin RNA (shRNA) expression vector driven by the U6 small nuclear RNA promoter, which can significantly suppress the expression of mouse GPM6A mRNA. Real-time polymerase chain reaction (real-time PCR) and immunocytochemical analysis showed that expression of shRNA against GPM6A markedly reduced the expression of neuroectodermal-associated genes (OTX1, Lmx1b, En1, Pax2, Pax5, Sox1, Sox2, and Wnt1), and also the number of neural stem cells (NSC) derived from D3mshM6A cells compared to control vector-transfected mouse ES cells (D3m-Mock). Moreover, our results show a decrease in both the number of neuronal markers and the number of differentiating neuronal cells (cholinergic, catecholaminergic, and GABAergic neurons) from NSC in D3m-shM6A cells. Hence, our findings suggest that expression level of GPM6A is directly or indirectly associated with the differentiation of neurons derived from undifferentiated ES cells.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2-/-γc-/-) mouse model

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

Effect of tumor suppressor gene PTEN on the resistance to cisplatin in human ovarian cancer cell lines and related mechanisms

Purpose The aim of this study was to explore role of PTEN gene in chemosensitivity to cisplatin in human ovarian cancer cells and related mechanisms. Method A PTEN-targeted short hairpin RNA (shRNA) expression vector and a wild-type sense PTEN plasmid were constructed, human ovarian cisplatin-sensitive cancer cell line OV2008 and its resistant variant C13∗ cells were transfected with PTEN shRNA or wild-type PTEN plasmid, respectively, and cells were then treated with cisplatin. Next, AKT activity was regulated with co-transfection of antisense or sense AKT plasmid in OV2008 /PTENshRNA cells or C13∗/p-PTEN cells, respectively. Effects of transfection of above vectors on cell growth, apoptosis and expression of PTEN and AKT were evaluated. Results Expression of PTEN in OV2008 cells was significantly higher than that in C13∗ cells. Transfection of PTEN shRNA into OV2008 cells remarkably down-regulated expression of PTEN and up-regulated expression of phospho-AKT protein, with transfected cells being resistant to cisplatin. Overexpression of PTEN by transfection with sense PTEN obviously enhanced cisplatin-induced apoptosis of C13∗ cells. Furthermore, decreased AKT activity could increase cisplatin-induced apoptosis in OV2008/PTENshRNA cells; while, transfection of pcDNA3.1-AKT plasmid into C13∗/p-PTEN cells resulted in increased activity of AKT, with cisplatin-induced apoptosis being inhibited significantly. Conclusions PTEN might reverse chemoresistance to cisplatin in human ovarian cancer cells through inactivation of the PI3K/AKT cell survival pathway and may serve as a potential molecular target for the treatment of chemoresistant ovarian cancer.

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer

Background and aims:Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification...

Application of RNAi Technology to the Inhibition of Zebrafish GtHα, FSHβ, and LHβ Expression and to Functional Analyses

Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.

Targeted inhibition of platelet-derived growth factor receptor-β subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats

The activation of hepatic stellate cells (HSCs) is the key event of the pathogenesis of hepatic fibrosis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs, and PDGF receptor-beta subunit (PDGFR-beta) is required for the proliferation of HSCs induced by PDGF. In this study, a high gene-silencing-efficacy PDGFR-beta small interference RNA (siRNA) was synthesized that could suppress the PDGFR-beta expression and inhibit the activation and proliferation but could not induce the apoptosis of HSCs in vitro. To avoid the side effect of nonspecific interference of PDGFR-beta, we constructed an HSCs-specific short hairpin RNA (shRNA) expression plasmid in which PDGFR-beta shRNA was driven by a glial fibrillary acidic protein (GFAP) promoter. The double-staining immunofluorescence examination indicated that GFAP promoter could target the transgene expression into HSCs in carbon tetrachloride induced acute injured rat's liver and bile duct ligation (BDL)-induced chronic injured rat's liver. Furthermore, HSCs-specific PDGFR-beta shRNA could relieve liver injury and hepatic fibrosis in the rat's model induced by BDL. This study demonstrates that PDGFR-beta siRNA may be presented as an antifibrogenic agent. The application of HSCs-specific RNA interference induced by the GFAP promoter might supply a new powerful tool for cell-specific gene therapy of hepatic fibrogenesis.

Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.

TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome‐associated protein NA14

TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjögren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.