CMV Promoter Research Articles

Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences.

The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single‐molecule‐based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway® system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5′‐UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5′‐UTR.

Functions of the Heterologous Intron-Derived Fragments Intra and Extra Factor IX-cDNA Coding Region on the Human Factor IX Expression in HepG2 and Hek-293T Cells.

BackgroundHuman FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3′-end formation of mRNAs are required.ObjectivesWe aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells.Materials and MethodsHepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells.ResultsOur data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compared to other constructs in Hek-293 cells. In HepG2 cells, the rABE in combination with CMVp in context of intron-less plasmid induced an increase in total expression of hFIX protein dramatically; ranging from 2.3 to 40 folds increase compared to other constructs. The rABE in combination with CMVp in the hBG intron-I, hBG intron-II, and hBG intron-I,II containing plasmids induced 3.7, 2, and 1.6-fold increase in the total expression of hFIX protein, respectively. The presence of both hBG intronic sequences within the hFIX-cDNA induced a higher secretion level of hFIX than either intron-I or II alone and provided correctly spliced hFIX transcripts in HepG2 and kidney cell lines. The intron-less construct with or without rABE induced the highest hFIX mRNA levels in HepG2 and Hek-293T cells respectively compared to other constructs.ConclusionsThe embryonic kidney cells in addition to the differentiated hepatic cell lines could be successfully targeted by plasmid vectors. The intron-less and hBG intron-I containing plasmids represent a particular interest in producing recombinant hFIX in Hek-293T cells. The synergistic function on the hFIX expression that was achieved by combining the CMVp with the liver-specific rABE would be a useful approach for future designing of the expression cassettes for hepatocyte-mediated gene expression in hemophilia B.

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing

Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.

Linkage disequilibrium and functional analysis of PRE1 insertion together with SNPs in the promoter region of IGFBP7 gene in different pig breeds.

Polymorphisms in regions upstream of transcription initiation site may modify the transcriptional activity of target genes by changing promoter activity. This study aims to determine whether or not polymorphisms at porcine IGFBP7 promoter region affect gene expression. In this study, eight SNPs and one PRE1 insertion in this region were first confirmed. The PRE1 insertion was widespread in 20 Chinese indigenous breeds, but was not observed in three commercial breeds. A perfect linkage disequilibrium, consisting of six of those SNPs and a PRE1, was observed with two haplotypes (h1 and h2) in five pig breeds. The h1 haplotype had an overwhelming superiority distribution in Large White, Landrace, and Bama mini-pig; in turn, the h2 only existed in the PRE1 presence breeds. As the haplotypes and PRE1 were located at gene promoter regions, we further investigated the transfection of plasmids with three different fragments of IGFBP-7 promoter region (H1, H2, RF). The CMV promoter of the pEGFP-N1 was substituted by these three different fragments, respectively. Different transcriptional and translational activities of EGFP in PK-15 cells were observed in these three recombinant plasmids by quantitative real-time PCR and flow cytometric analysis. The results indicated that H1 had the higher transcriptional and translational activities of EGFP as compared to the H2 (P < 0.05, P < 0.05). As compared to the RF group, EGFP mRNA expression level was significantly higher in H1 groups (P < 0.05). The IGFBP-7 promoter polymorphisms detected in this study may be important functional variants and potential genetic markers for pig population genetic study.

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.

Inhibition of experimental autoimmune encephalomyelitis by tolerance-promoting DNA vaccination focused to dendritic cells.

In this study we analysed the effects of prophylactic biolistic DNA vaccination with plasmids encoding the encephalitogenic protein myelin oligodendrocyte glycoprotein (MOG) on the severity of a subsequently MOGp35-55-induced EAE and on the underlying immune response. We compared the outcome of vaccination with MOG-encoding plasmids alone or in combination with vectors encoding the regulatory cytokines IL-10 and TGF-ß1, respectively. MOG expression was restricted to skin dendritic cells (DCs) by the use of the DC-specific promoter of the fascin1 gene (pFscn-MOG). For comparison, the strong and ubiquitously active CMV promoter was employed (pCMV-MOG), which allows MOG expression in all transfected cells. Expression of IL-10 and TGF-ß1 was controlled by the CMV promoter to yield maximal synthesis (pCMV-IL10, pCMV-TGFß). Co-application of pFscn-MOG and pCMV-IL10 significantly ameliorated EAE pathology, while vaccination with pCMV-MOG plus pCMV-IL10 did not affect EAE outcome. In contrast, vaccination with either of the two MOG-encoding plasmids in combination with pCMV-TGFß significantly attenuated the clinical EAE symptoms. Mechanistically, we observed diminished infiltration of Th17 and Th1 cells as well as macrophages/DCs into the CNS, which correlated with decreased MOGp35-55-specific production of IL-17 and IFN-ϫ by spleen cells and reduced peptide-specific T cell proliferation. Our findings suggest deletion of or anergy induction in MOG-specific CD4+ T cells by the suppressive vaccination platform employed. MOG expression driven by the DC-specific fascin1 promoter yielded similar inhibitory effects on EAE progression as the ubiquitously active viral CMV promoter, when coapplying pCMV-TGFß. Our finding that pCMV-IL10 promoted tolerogenic effects only, when coapplied with pFscn-MOG, but not pCMV-MOG suggests that IL-10 affected only directly transfected DCs (pFscn-MOG), but not neighbouring DCs that engulfed MOG-containing vesicles derived from transfected keratinocytes (pCMV-MOG). Thus, due to its DC-restricted expression, the fascin1 promoter might be an interesting alternative to ubiquitously expressed promoters for vaccination strategies.

Mitochondrial transgene expression via an artificial mitochondrial DNA vector in cells from a patient with a mitochondrial disease

To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV-mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO-Porter, a liposome-based carrier for delivering a cargo to mitochondria via membrane fusion. We report herein that mitochondrial transfection using the MITO-Porter results in mitochondrial transgene expression in G625A fibroblasts obtained from a patient with a mitochondrial disease. We investigated the effect of promoters and the basic structure of pCMV-mtLuc (CGG) on gene expression efficiency, and were able to construct a high performance mitochondrial DNA vector, pCMV-mtLuc (CGG) [hND4] that contains a human mitochondrial endogenous gene. We also constructed an RP/KALA-MITO-Porter composed of the KALA peptide (cell-penetrating peptide) with a mitochondrial RNA aptamer to enhance cellular uptake and mitochondrial targeting. Finally, the mitochondrial transfection of pCMV-mtLuc (CGG) [hND4] in G625A fibroblasts using the RP/KALA-MITO-Porter resulted in strong mitochondrial transgene expression.

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury

PurposeGene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome.MethodsDouble-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types.ResultsSuccessful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve.ConclusionsDamaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

201 Spermatogonia Transplantation in the Chicken

Spermatogonia are the precursors of male germ cells. They are a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the spermatogonia from male donor’s testes, transformation, and transplantation of donor cells into the sterilized recipient’s testes. The transplanted spermatogonia subsequently differentiate into male sex cells (sperm). The aim of this study was to optimize the individual stages of donor spermatogonia transplantation into the recipient’s testes to increase the effectiveness of spermatogenesis recovery. In the first stage, the spermatogenesis in male chicken was examined to determine the optimal age for isolation of spermatogonia from testes. Histological examinations of male chicken testes (n = 80 birds) were done for 8 age categories, from 1 week to 3 months. It was found that under the age of 4 weeks, the cell population in the seminiferous tubules of male chickens was represented mainly by Sertoli cells and spermatogonia. Maximum percentage of spermatogonia was 69 ± 3% at 4 weeks. At the next stage, a culture of spermatogonia was obtained. Testes of 3-week-old male chickens were used. Separation of the spermatogonia from other types of cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population was established by transfer (3 times) of the supernatant containing unattached cells after 24 h of cultivation into a new culture dish for further cultivation. The cell population is represented mainly by the spermatogonia (89 ± 3%). The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the spermatogonia. The efficiency of spermatogonia infection with lentiviral particles (TU/mL = 2.5 × 108) was 65 ± 2%. After transformation, spermatogonia were introduced into the testes of busulfan-sterilized recipients. The optimal concentration of busulfan treatment after series of experiments from 40 to 100 mg/kg was determined. The effective dose for the removal of own spermatogenic cells was revealed at a concentration of 80 mg/kg of live weight. With complete elimination of other types of spermatogenic cells, the number of Sertoli cells and spermatogonia in the testicle tubules decreased by 39 ± 2% and 98 ± 1%, respectively, compared with the control group. The efficiency of spermatogenesis recovery was assessed based on sperm analysis that was obtained from male recipients (n = 5 birds) 4 months after the introduction of donor cells using PCR. The presence of recombinant DNA (ZsGreen) in recipients’ sperm was shown. Thus, our results indicate the prospect of using spermatogonia as a genetic material for the production of transgenic poultry. Study was supported by the Russian Science Foundation (Project no.16-16-10059).

200 Optimization of Individual Stages of Chicken Transgenesis to Increase Efficiency

Primordial germ cells (PGC) are the precursors of male and female progenitor cells. The cells are considered a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the PGC from chick donor embryos, transformation of the cells, and injection into the dorsal aorta of recipient embryos. After injection, the PGC are involved in the process of embryo development and differentiate into male or female sex cells. The aim of the research was to optimize the individual stages of this technology to increase the efficiency of transgenesis. The PGC were extracted from embryo gonads at stage 26 to 27 (H&amp;H) using the trypsinization process. The trypsin concentration and incubation time were determined experimentally. Treatment of chick embryos with a 0.05% trypsin solution for 5 min was optimal for obtaining culture of embryonic cells. Separation of the PGC from other types of embryonic cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population for further cultivation was established by transfer (twice) of the supernatant containing unattached cells after 1 h of cultivation in a new culture dish. The cell population is represented mainly by the PGC (81 ± 4%). Additional purification of the PGC from other cell types using magnetic-activated cell sorting (MACS) increased the proportion of these cells up to 93 ± 2%. The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the PGC. The efficiency of infection of PGC with lentiviral particles (TU/mL = 2.5 × 108) was 70 ± 3%. The transformed cells were injected into the dorsal aorta of recipient embryos on Day 2.5 (n = 80). Before injecting donor PGC, recipient embryos were treated with busulfan to remove the endogenous PGC. The optimal dose of busulfan was selected experimentally. A series of experiments introducing busulfan in concentrations from 50 to 250 μg into chick embryos at 24 h of incubation showed that the optimal dose was 100 μg/embryo. The efficacy of colonization of gonads with donor PGC was assessed on Day-10 embryos (n = 32) and 4-week-old hatched chickens (n = 12). Cells from gonads were studied using fluorescence microscopy, fluorescence-activated cell sorting (FACS) and qPCR. The presence of fluorescent cells in the gonads of recipients was established in both embryos and hatched chickens. The relative number of the recombinant DNA copies and the relative level of expression were confirmed by qPCR. The FACS analysis of sex cells isolated from gonads of recipients showed that the percentage of transformed germ cells reached 55.8% in females (n = 5) and 31.9% in males (n = 7). Thus, the effectiveness of poultry transgenesis can be enhanced by preparation of donor PGC for injection into embryo recipients and elimination of endogenous PGC in recipients. Both the purification of PGC from other cell types based on adhesive capacity as well as treatment of embryo recipients at 24 h incubation with busulfan (100 μg/embryo) increased the effectiveness of transgenesis. Study supported by the RSF within project No. 16-16-10059.

Protective efficacy of an IL-12-expressing baculoviral malaria vaccine.

Interleukin-12 (IL-12) plays an important role in antigen-specific adaptive immunity against Plasmodium sporozoites, and this requirement allows for a new approach to developing an effective malaria vaccine. In this study, we examined whether IL-12 could enhance protective efficacy of a baculovirus-based malaria vaccine. For this aim, a baculoviral vector expressing murine IL-12 (mIL-12) under the control of CMV promoter (BES-mIL-12-Spider) and a baculoviral vector expressing Plasmodium falciparum circumsporozoite protein (PfCSP) with post-transcriptional regulatory element of woodchuck hepatitis virus (BDES-sPfCSP2-WPRE-Spider) were generated. BES-mIL-12-Spider produced bioactive IL-12 which activates splenocytes, resulting in induction of IFN-γ. When co-immunized with BES-mIL-12-Spider and BDES-sPfCSP2-WPRE-Spider, the mouse number for high IgG2a/IgG1 ratios and the geometric mean in this group were both increased as compared with those of the other groups, indicating a shift towards a Th1-type response following immunization with BES-mIL-12-Spider. Finally, immunization with BDES-sPfCSP2-WPRE-Spider plus BES-mIL-12-Spider had a higher protective efficacy (73%) than immunization with BDES-sPfCSP2-WPRE-Spider alone (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. These results suggest that co-administration of IL-12 expressing baculoviral vector, instead of IL-12 cDNA, with viral-vectored vaccines provides a new feasible vaccine platform to enhance Th1-type cellular immune responses against Plasmodium parasites.

Establishment of a platform for molecular and immunological characterization of the RNA-dependent-RNA-polymerase NS5B of an Egyptian HCV isolate.

The present work aimed at establishing a platform to enable frequent characterization of the HCV RNA-dependent-RNA-polymerase from Egyptian clinical isolates. Subjecting amplified HCV-NS5B coding gene from Egyptian patient's serum to sequencing, multiple alignment, and phylogenetic analysis confirmed its subtype 4a origin. Nucleotide sequence analysis revealed presence of an additional start codon at the beginning of the NS5B gene. Peptide sequence alignment demonstrated presence of unique amino acid residues in our 4a-NS5B sequence distinct from the JFH-1-NS5B sequence as well as unique amino acids compared to other genotypes. The distinct molecular structure of the herein characterized 4a-NS5B from the 2a-JFH-1-NS5B was further demonstrated both in the built 3D models and the Ramachandran plots corresponding to each structure. Both the unique amino acid residues and 3D structure of the 4a-NS5B may influence both genotype 4a replication rate and response to therapy in comparison to other genotypes. Many resistance mutations to polymerase inhibitors were found both in ours and other genotypes' sequences. The presence of the required amino acid motifs for the RNA dependent RNA polymerase activity encouraged to clone the NS5B570-encoding sequence downstream CMV promotor in a mammalian expression vector. Such construct was used for both prokaryotic expression in bacteria and for DNA immunization. Successful mammalian expression and induction of specific immune response were demonstrated by ELISA and Western blotting. The potential of both the raised antibodies and the expressed NS5B to differentiate between HCV-infected and control human sera were demonstrated which reflect their diagnostic value.

Functional characterisation of filamentous actin probe expression in neuronal cells.

Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

Non-invasive detection of bladder cancer via expression-targeted gene delivery.

Because of the time and expense associated with the procedures and possible distress to the patient, cystoscopy or other imaging techniques are typically not used for bladder cancer detection before symptoms become present. Alternatively, commercial assays for urinary tumor markers exist but are marred by low sensitivity and high cost. There is a need for a simple and sensitive means of tumor detection, such as via the analysis of urine. Plasmids encoding the secretable reporter Gaussia Luciferase (G.LUC), under the control of cmv, cox2 or opn promoters, were delivered via polyethylenimine into bladder tumor cells in culture and into the bladders of mice. Expression profiles of the reporter were recorded, the optimal times for reporter detection were determined and the relationship of reporter expression with tumor size was calculated. In vitro results showed that both the cox2 and opn promoters can drive significant expression of G.LUC in bladder carcinoma cells in a targeted fashion. In vivo results demonstrated that the cox2 promoter caused expression of G.LUC at detectable levels in the urine, with local signal maxima occurring at 48 and 72 h post-transfection. G.LUC levels in the urine had a 24-h periodicity, with the periodicity partly being the result of an agent secreted by tumor cells that served to mask the luciferase signal. Having shown tumor specificity and having been calibrated with respect to circadian expression patterns, the detection system shows great promise for future investigation of tumor presence both in the urinary bladder and other models of cancer.

Gene delivery of hypoxia-inducible VEGF targeting collagen effectively improves cardiac function after myocardial infarction

Vascular endothelial growth factor (VEGF) plays important roles in improvement of cardiac function following myocardial infarction (MI). However, the lack of a steerable delivery system of VEGF targeting the infarcted myocardium reduces the therapeutic efficacy and safety. Here, we constructed a series of lentiviral vector systems which could express a fusion protein consisted of a collagen-binding domain (CBD) and hVEGF (CBDhVEGF), under the control of 5HRE-hCMVmp (5HRE), the hypoxia-inducible promoter consists of five copies of the hypoxia-responsive element (HRE) and a human cytomegalovirus minimal promoter (hCMVmp). We demonstrated that 5HRE has the comparable ability to strongly drive CBDhVEGF under hypoxic condition as the ubiquitous CMV promoter, but it can hardly drive target gene under normoxic condition. 5HRE-drived CBDhVEGF specifically bound to type I collagen and significantly promoted the viability of HUVEC cells. Moreover, after injection of lentivirus into heart of mouse with MI, CBDhVEGF was mainly retained in infarcted myocardium where containing rich collagen and significantly improved angiogenesis and cardiac function when compared with hVEGF. Moreover, CBDhVEGF mediated by lentivirus has little leakage from infarcted zone into blood than hVEGF. Taken together, our results indicate that 5HRE-CBDhVEGF lentiviral vector system could improve cardiac function in the collagen-targeting and hypoxia-inducible manners.

Construction of weak immunogenicity virus vector with super antigen staphylococcal enterotoxin A gene

Objective To construct a weak immunogenicity lentiviral vector expressing the staphylococcal enterotoxin A (SEA) gene in order to exert a potent antitumor effect on activated T cells. Methods The human telomerase reverse transcriptase (hTERT)-CMV promoter sequence of 851 bp and the SEA gene of 774 bp were obtained by polymerase chain reaction (PCR). The target genes were constructed into lentiviral vector plasmids. After sequencing, they were transferred into DH5α competent cells and amplified. Viral supernatants were collected by co-transfection of 293FT cells with shuttle plasmids and packaging plasmids, and virus titer was determined by fluorescence titer. Different concentrations of virus supernatant were transfected into BIU-87 cell line. The target gene was sequenced and the expression of SEA gene was detected by Western blotting. Results The size of the vector was 10 458 bp, and the gene sequence was correct. No obvious mutation was found. The titer of the virus titer was (4.30±2.00)×109 TU/ml. PCR product sequencing of the target gene confirmed that the target gene was correct, the total RNA were 742、803、726 mg/L respectively by spectrophotometric assay. Western blotting revealed that SEA gene was successfully expressed. Conclusion The weak immunogenicity virus carrying the superantigen SEA gene was successfully constructed. Key words: Lentivirus; Staphylococcal enterotoxin A; Tumor; Super antigen

Expression of Thiaminase in Zebrafish (Danio rerio) is Lethal and Has Implications for Use as a Biocontainment Strategy in Aquaculture and Invasive Species.

As the world increasingly relies on aquaculture operations to meet rising seafood demands, reliable biocontainment measures for farmed fish stocks are desired to minimize ecological impacts arising from interactions of cultured fish with wild populations. One possible biocontainment strategy is to induce a dietary dependence on a vitamin, such as thiamine (vitamin B1), required for survival. Fish expressing thiaminase (an enzyme that degrades thiamine) within a confined aquaculture facility could receive supplemental thiamine to allow survival and normal growth, whereas escapees lacking this dietary rescue would die from thiamine deficiency. To test the concept and efficacy of such a dietary dependency system (for potential future use in larger aquaculture species), we expressed thiaminase in zebrafish as a test model. We drove the expression of thiaminase under the strong ubiquitous and constitutive control of the CMV promoter which resulted in non-viable fish, indicating that the thiaminase sequence kills fish. However, the CMV promoter is too strong to allow conditional survival since the lethality could not be rescued by exogenous thiamine provided as a supplement to typical food. In addition, microinjection of 0.5pg of thiaminase mRNA in zebrafish embryos at the one-cell stage resulted in 50% larval mortality at 5days post-fertilization (dpf), which was partially rescued by thiamine supplementation. Evaluating the efficacy of biocontainment strategies helps assess which methods can reliably prevent ecological impacts arising from breaches in physical containment systems that release engineered organisms to nature, and consequently provides critical information for use in regulatory risk assessment processes.

Glial fibrillary acidic protein promoter determines transgene expression in satellite glial cells following intraganglionic adeno-associated virus delivery in adult rats.

Recombinant adeno-associated viral (AAV)-mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Müller glia-prone transduction. Titer-matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6-EGFP and AAVshH10-EGFP driven by a GFAP promoter (AAV6-GFAP-EGFP and AAVshH10-GFAP-EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6- and AAVshH10-GFAP-EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRG resulted in a similar in vivo transduction profile to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.

A Novel Retinal Ganglion Cell Promoter for Utility in AAV Vectors

Significant advances in gene therapy have enabled exploration of therapies for inherited retinal disorders, many of which are in preclinical development or clinical evaluation. Gene therapy for retinal conditions has led the way in this growing field. The loss of retinal ganglion cells (RGCs) is a hallmark of a number of retinal disorders. As the field matures innovations that aid in refining therapies and optimizing efficacy are in demand. Gene therapies under development for RGC-related disorders, when delivered with recombinant adeno associated vectors (AAV), have typically been expressed from ubiquitous promoter sequences. Here we describe how a novel promoter from the murine Nefh gene was selected to drive transgene expression in RGCs. The Nefh promoter, in an AAV2/2 vector, was shown to drive preferential EGFP expression in murine RGCs in vivo following intravitreal injection. In contrast, EGFP expression from a CMV promoter was observed not only in RGCs, but throughout the inner nuclear layer and in amacrine cells located within the ganglion cell layer (GCL). Of note, the Nefh promoter sequence is sufficiently compact to be readily accommodated in AAV vectors, where transgene size represents a significant constraint. Moreover, this promoter should in principle provide a more targeted and potentially safer alternative for RGC-directed gene therapies.

RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.