Exogenous Protein Expression Research Articles

Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging.

Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

Gene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoter.

We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.

The Relationship between Insect Resistance and Tree Age of Transgenic Triploid Populus tomentosa Plants.

To explore the stability of insect resistance during the development of transgenic insect-resistant trees, this study investigated how insect resistance changes as transgenic trees age. We selected 19 transgenic insect-resistant triploid Populus tomentosa lines as plant material. The presence of exogenous genes and Cry1Ac protein expression were verified using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. The toxicity for Clostera anachoreta and Lymantria dispar was evaluated by feeding fresh leaves to first instar larvae after the trees were planted in the field for 2 years and after the sixth year. Results of PCR showed that the exogenous genes had a long-term presence in the poplar genome. ELISA analyses showed significant differences existed on the 6-year-old transgenic lines. The insect-feeding experiment demonstrated significant differences in the mortality rates of C. anachoreta and L. dispar among different transgenic lines. The average corrected mortality rates of C. anachoreta and L. dispar ranged from 5.6–98.7% to 35.4–7.2% respectively. The larval mortality rates differed significantly between the lines at different ages. Up to 52.6% of 1-year-old transgenic lines and 42.1% of 2-year-old transgenic lines caused C. anachoreta larval mortality rates to exceed 80%, whereas only 26.3% of the 6-year-old transgenic lines. The mortality rates of L. dispar exhibited the same trend: 89.5% of 1-year-old transgenic lines and 84.2% of 2-year-old transgenic lines caused L. dispar larval mortality rates to exceed 80%; this number decreased to 63.2% for the 6-year-old plants. The proportion of 6-year-old trees with over 80% larval mortality rates was clearly lower than that of the younger trees. The death distribution of C. anachoreta in different developmental stages also showed the larvae that fed on the leaves of 1-year-old trees were killed mostly during L1 and L2 stages, whereas the proportion of larvae that died in L3 and L4 stages was significantly increased when fed on leaves of 6-year-old trees. Results of correlation analysis showed there was a significant correlation between the larvae mortality rates of trees at different ages, as well as between Cry1Ac protein contents and larvae mortality rates of 6-year-old trees.

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-β (Aβ) peptide.

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-β (Aβ) peptide.

Characterisation of a stably integrated expression system for exogenous protein expression in DT40

The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV, CBA or CAG, and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful experimental tool for future DT40 experiments.

Design, Assembly, Production, and Transfection of Synthetic Modified mRNA.

Proteins are drivers of cell functions and are targets of many therapies. Exogenous protein expression techniques, therefore, have been essential for research and medicine. The most common method for exogenous protein expression relies on DNA-based viral or non-viral vectors. However, DNA-based vectors have the potential to integrate into the host genome and cause permanent mutations. RNA-based vectors solve this shortcoming. In particular, synthetic modified mRNA provides non-viral, integration-free, zero-footprint method for expressing proteins. Modified mRNA can direct cell fate specification and cellular reprogramming faster and more efficiently than other methods. Furthermore, when simultaneously express multiple different proteins, mRNA vectors allow for greater flexibility and control over stoichiometric ratios, dose titrations, and complete silencing of expressions. Additionally, modified mRNAs have been shown to be viable and safe as therapeutic agents for gene therapy and vaccine, providing an alternative approach to address diseases. Despite these advantages, technical challenge, mRNA instability, and host immunogenicity have caused significant barriers to widespread use of this technology. The comprehensive method presented here addresses all of these shortcomings. This stepwise protocol describes every step necessary for the synthesis of modified mRNA from any coding DNA sequence of interest. The meticulously detailed protocol enables the users to make alterations to each component of modified mRNA for even more significant customization, allowing the researchers to apply this technology to a wide range of uses. This non-cytotoxic synthetic modified mRNA can be used for protein expression, regulation of cell reprogramming or differentiation, and drug delivery.

Characterisation of a stably integrated expression system for exogenous protein expression in DT40

The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV, CBA or CAG, and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful experimental tool for future DT40 experiments.

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic.

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

TGF-β1 signaling pathway serves a role in HepG2 cell regulation by affecting the protein expression of PCNA, gankyrin, p115, XIAP and survivin.

The transforming growth factor-β (TGF-β) signaling pathway serves a key role in the pathogenesis of liver cancer. To investigate the association between TGF-β1 and the following proteins: Proliferating cell nuclear antigen (PCNA), gankyrin, general vesicular transport factor p115 (p115), X-linked inhibitor of apoptosis protein (XIAP) and survivin, HepG2 liver cancer cells were transfected with small interfering RNA (siRNA) directed against TGF-β1, or were treated with exogenous TGF-β1. TGF-β1 protein expression levels were assessed at 72 and 96 h using western blotting, cell growth was evaluated using a Cell Counting kit-8 assay, and flow cytometry was used to examine cell cycle distribution and apoptosis. In addition, PCNA, gankyrin, p115, XIAP and survivin protein levels were evaluated using western blotting. TGF-β1 protein expression levels were decreased at 72 and 96 h following siRNA transfection, indicating that the siRNA against TGF-β1 was effective. In the TGF-β1-knockdown group, the HepG2 cells exhibited G1 or S-phase cell cycle arrest; therefore, the number of G2-phase cells was decreased, cell growth was inhibited and apoptotic peaks were observed. By contrast, no significant alteration in cell cycle distribution or apoptosis was observed in the cells treated with exogenous TGF-β1. In the exogenous TGF-β1 group, PCNA and XIAP protein expression levels were increased, whereas gankyrin, p115 and survivin protein expression was observed to be dependent on the duration of treatment. By contrast, PCNA, gankyrin, XIAP and survivin protein expression decreased following TGF-β1 knockdown; however, p115 protein expression increased. In conclusion, the TGF-β1 signaling pathway may affect cell growth, cell cycle distribution and apoptosis through the regulation of PCNA, gankyrin, p115, XIAP and survivin protein expression in liver cancer. The results of the present study may improve the current understanding of the role of the TGF-β signaling pathway during the pathogenesis of liver cancer.

Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. “adenovirus driven NPT2a-EGFP and endogenous NHE3 protein”, “adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein” and “adenovirus driven AQP2-EGFP and endogenous AQP2 protein”. Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

Hydrophobically modified polyethylenimine-based ternary complexes for targeting brain tumor: stability, in vitro and in vivo studies

Hydrophobic modification of low molecular weight polyethylenimine (PEI 2 kDa) by cholic acid (ChA) was done to obtain PEI2-ChA. The nanoplexes of PEI2-ChA with gWIZ-GFP demonstrated increase transfection efficiency (∼27%) in NT8e cell lines. The cell-cycle analysis of NT8e cells (p53 mutant) treated with transferrin containing nanoplexes showed increased apoptosis of cells. In vitro protein expression revealed expression of exogenous p53 protein. In vivo imaging of mice showed localized signal for GFP protein in brain region. The tumors of mice treated with transferrin containing nanoplexes of PEI2-ChA were ∼5 times smaller in size than the tumor of untreated animals.

Production of transgenic chickens constitutively expressing human erythropoietin (hEPO): Problems with uncontrollable overexpression of hEPO gene

The chicken is a promising candidate as a bioreactor for the economical mass production of human therapeutic proteins. Here, we report the successful generation of transgenic chickens that produce high concentrations of human erythropoietin (hEPO) in the blood. Using a Moloney murine leukemia virus (MoMLV)-based pseudotyped retrovirus vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G), the hEPO gene under the control of cytomegalovirus (CMV) promoter was introduced to the blastoderm of freshly laid chicken eggs (stage X). Out of 200 injected eggs, 12 chicks were hatched after 21 days of incubation, and all of the G0 hatched chicks expressed the vector-encoded hEPO gene. One of the G0 roosters successfully transmitted the hEPO gene to its G1 progeny by crossing with non-transgenic hens. The concentration of hEPO protein in the chicken blood serum was as high as 90 μg/mL. Although humans and chickens belong to different classes of the phylogenetic tree, human EPO caused devastating problems in transgenic chickens, including sudden death, polycythemia, vasodilation, and so on, which may be due to the uncontrolled constitutive expression of exogenous protein in the chicken body. Despite many disorders, however, we were able to generate chicks of G2 generation sired by a rooster of G1 generation confirming successful establishment of a new line of transgenic chicken characterized by high expression of the hEPO gene. With these chickens, we believe that studies on the evaluating the possibilities of the transgenic animal-mediated bio-pharming and on the hEPO-induced physiological side effects will be greatly facilitated.

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line, Pv11.

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13)

A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells.

Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species.

Characterization of actin and tubulin promoters from two sap-sucking pests, Nilaparvata lugens (Stål) and Nephotettix cincticeps (Uhler)

The brown planthopper, Nilaparvata lugens (N. lugens, Hemiptera: Delphacidae), and the green rice leafhopper, Nephotettix cincticeps (N. cincticeps, Hemiptera: Cicadellidae), two sap-sucking feeders, have caused many destructive agricultural disasters in East Asia, as they can bring diseases like ‘hopper burn’ and transmit plant viruses. Recently, continuously-cultured cell lines from both insects have been reported. However, exogenous protein expression systems have not yet been established. Here, we identified thirteen tubulin genes and three actin genes from N. lugens, and one tubulin gene and two actin genes from N. cincticeps. Furthermore, putative promoter regions of these genes were analyzed by bioinformatic approaches and 5′-RACE assay, and the promoter strength was evaluated by driving the enhanced green fluorescent protein expression in three insect cell lines, S2, Sf9, and BmN. Finally, we identified three effective promoters (Nl_αTub1 promoter, Nl_act3 promoter, and Nc_act1 promoter) among all candidates we screened. The Nc_act1 promoter showed the strongest activity, while the Nl_αTub1 promoter only worked in S2 cells. In conclusion, we identified and functionally characterized three native promoters from N. lugens and N.cincticeps, which would facilitate the establishment of exogenous protein expression systems suitable for these two insect pests.

Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization.

Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research.