EGFP Expression Research Articles

Evaluation of two methods to isolate Schwann cells from murine sciatic nerve

BackgroundSchwann cells (SC) and macrophages play key roles in the response to peripheral nerve injury (PNI). Accurate isolation of such cells is essential for further analyses that can lead to better understanding of the repair process after PNI. Separation of live SC from the injury site without culture enrichment is necessary for targeted gene expression analysis. New methodsTwo flow cytometric techniques are presented for rapid enrichment of live SC and macrophages from injured murine peripheral nerve without the need for culture. ResultsSC were isolated by fluorescent activated cell sorting (FACS) using transgenic expression of eGFP in SC, or by exclusion of other cell types collected from the injury site. Comparison with existing method(s)Gene expression analyses of peripheral nerve repair have commonly used whole nerve lysates. Isolating SC allows more accurate understanding of their specific role in repair. SC are commonly enriched from nerve by culture, however this changes gene expression patterns and limits the utility for transcriptomic analysis. The surface marker p75-NTR has variable expression in different SC phenotypes and during the course of injury and repair. Using p75-NTR for SC isolation might enrich only a subset of SC. More stably expressed lineage markers for SC are intracellular and not suitable for sorting for gene expression. The methods used here avoid the requirement for surface marker labeling of SC. ConclusionGene expression analysis of sorted cells from both methods showed successful enrichment of SC. Lineage markers such as Map1b, p75-NTR and S100b were enriched in the sorted SC population. SC sorting by eGFP expression showed improved enrichment, particularly of mature myelinating genes, although this could represent sampling of a subset of SC.

Foamy Virus Based Vectors for CAR-T Cell Development

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

Transplanted Vasoactive Intestinal Peptide-Secreting Donor Plasmacytoid Dendritic Cells Migrate to Lymphoid Organs and GvHD Target Organs

Introduction: Donor plasmacytoid dendritic cells (pDCs) have important roles in the pathogenesis of graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vasoactive intestinal peptide (VIP), a potent anti-inflammatory neuropeptide with pleiotropic effects, has shown promise in managing GvHD through induction of regulatory dendritic cells (Chorny, Blood 2006) and regulatory T cells (Delgado, JLB 2005). Our previous data showed that transplantation of hematopoietic stem cells (HSC) lacking VIP expression in enhanced Th1 polarization and antiviral immunity (Li, JI 2011). Whether VIP produced by pDCs plays a key role in preventing GvHD is the focus of the current study. Method: A reductive allo-HSCT model was established by transplanting purified populations of 5 x 103 hematopoietic stem cells (HSC), 5 x 104 pDCs and 106 T cells from B6 (H-2Kb) to MHC mismatched B10.BR (H-2Kk) recipients. Mice received either VIP knockout pDCs (experimental group) or wild-type pDCs (control). Survival and clinical GvHD scores were monitored for at least 60 days. Serum pro-inflammatory cytokines were measured by luminex on day 3 and day 8. Additional recipients were transplanted for interim analyses of GvHD pathology in histological sections of small intestine and colon on day 8 and day 15. To investigate pDC migration and proliferation status, 5 x 104 luciferase-expressing pDCs, together with 5 x 103 WT B6 HSC, were transplanted to MHC mismatched Balb/c (H2kd) recipients. Bioluminescent imaging was performed on day 14. We used mice carrying an eGFP reporter linked to a VIP promoter to measure VIP production by donor cells in vivo. The construct has the eGFP reporter gene, followed by a polyadenylation sequence, inserted into BAC clone RP23-25A8 at the initiating ATG codon of the first coding exon of the VIP gene, so that eGFP expression is driven by the regulatory sequences of the BAC gene. The BAC is inserted randomly in the genome. We used serial breeding to establish the eGFP-VIP reporter in B6 mice. Bone marrow pDCs were sorted from transgenic eGFP-VIP B6 mice and used in transplant experiments, allowing VIP gene activity from donor-derived pDCs to be detected as GFP expression using confocal microscopy. Results: Compared to recipients of WT pDCs, recipients of VIP-KO pDCs have a significantly lower survival rate with increased GvHD clinical score (Figure 1; *p<0.05). Transplant recipients of VIP-KO donor pDC had higher serum pro-inflammatory cytokine levels on day 3 and day 8 respectively. (Figure 2). At 2 weeks post-transplant donor pDCs migrated primarily to the spleen, with limited presence of donor pDCs in the gastrointestinal tract and lung (Figure 3). Compared to mice transplanted with WT pDCs, recipients of VIP-KO pDCs had increased donor T cell migration to the lung and paw, which are chronic GvHD target organs after two weeks. (Figure 4; *p<0.05). When stimulated with IFN-g, eGFP-VIP transgenic pDCs have activation of the VIP gene promoter in vitro, as detected using eGFP (Figure 5). Confocal microscopy data showed that pDCs have activated the VIP promoter and secreted more VIP following stimulation by IFN-g, consistent with VIP up-regulation by the inflammatory mileu in allo-transplant recipients. Conclusions: These data show that lack of VIP secretion by donor pDCs resulted in lower survival rates and higher GvHD scores in allo-BMT recipients. Recipients of VIP KO donor pDC had increased donor T cell proliferation in GvHD target organs and increased GvHD clinical signs. The major site of donor pDCs migration in recipients is spleen, suggesting that pDCs may interact with other lymphoid cells, such as T cells, inducing a more tolerogenic environment. Donor pDCs were also found, to a lesser extent, in the lung and gastrointestinal tract, which are both GvHD target organs. Thus, pDCs may protect recipients from GvHD by limiting the activity of alloreactive T cells at these sites. The mechanism by which VIP-secreting donor pDCs regulate GvHD will be discussed. Disclosures Waller: Amgen: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Other: Travel expenses, Research Funding; Cerus Corporation: Other: Stock, Patents & Royalties; Chimerix: Other: Stock; Cambium Oncology: Patents & Royalties: Patents, royalties or other intellectual property ; Kalytera: Consultancy.

Application of internal control based on recombinant retroviral particles for increasing the PCR assay reliability

Molecular diagnostic tests based on PCR preceded by reverse transcription (RT-PCR) are now used commonly for the detection of viral pathogens with RNA genomes. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by inhibition or inefficient extraction. In the present study a strategy to produce a new type of internal control for RT-PCR based on recombinant retroviral particles is described. Cell clones stably producing retroviral particles were established by transfecting GP+env-AM12 packaging cells with constructed MoMuLV-derived retroviral vector pLneo/gfp and subsequent cultivation on selective medium with G418. Theegfpgene was used as a target for primers and hybridization probe design for real-time RT-PCR assay and as a marker for flow cytometry analysis of eGFP expression by transfected cells. The developed internal control is stable and ribonuclease resistant, economical to produce, noninfectious and safe for routine use. It closely mimics the natural virus and could be successfully used to monitor all the stages of RT-PCR, including nucleic acid extraction, RNA reverse transcription and amplification.

Extracellular DNA Containing (dG)n Motifs Penetrates into MCF7 Breast Cancer Cells, Induces the Adaptive Response, and Can Be Expressed.

Background Oxidized human DNA or plasmid DNAs containing human ribosomal genes can easily penetrate into the breast cancer cells MCF7 and stimulate the adaptive response induction. Plasmid DNA containing a CMV promoter, gene EGFP, and the insertion of the human ribosomal genes can be expressed. A hypothesis is proposed: these features of the ribosomal DNA are due to the presence of dGn motifs that are prone to oxidize. Methods Cells of MCF7 line were cultured with plasmids which contained a CMV promoter and gene of fluorescent protein EGFP. Genetic construction pEGFP-Gn contains pEGFP vector and a small insertion with dG11 and dG13 motifs that are inclined to oxidation. The accumulation of pEGFP and pEGFP-Gn in MCF7 (qPCR), the levels of ROS in the cells, the content of 8-oxodG in plasmids and cellular DNA (flow cytometry, immunoassay, and fluorescent microscopy), the expression of NOX4 and EGFP, the localization of NOX4 and EGFP in MCF7 (qPCR, flow cytometry, and fluorescent microscopy), and the levels of the cell DNA damage (comet assay) were analyzed. Results (dG)n insertions in the plasmid pEGFP increase the levels of ROS, the cell DNA oxidation and DNA damage, and the level of transfection of plasmid into the MCF7 cells. NOX4 participates in the oxidation of pEGFP-Gn and pEGFP. The expression of EGFP gene in MCF7 is significantly increased in case of pEGFP-Gn. Stimulation of ROS synthesis (H2O2 40 μM or 10 cGy IR) increases the level of expression of EGFP. Conclusions GC-rich DNA fragments containing dGn motifs that are inclined to oxidation penetrate into MCF7 cancer cells, stimulate the adaptive response, and can be expressed. This property of GC-rich cell-free DNA should be considered and/or could potentially be used in therapy of tumors.

Enhanced transgene expression using two β-globin MARs flanking expression cassettes in stably transfected CHO-K1 cells.

In this study, we systemically investigated the positions and orientations of matrix attachment regions (MARs) in expression vectors to fully explore the mechanism for improving transgene expression. We constructed 14 vectors that incorporated human β-globin MARs into pIRES-eGFP backbone vectors. The MARs flanked the eGFP expression cassette or promoter in a forward/reverse orientation. After stable transfection into CHO-K1 cells with these vectors, eGFP expression levels were increased significantly relative to that of the control vector (MAR-devoid) when two MARs flanking the expression cassette were incorporated, followed by those at the 5' site (upstream of the promoter). Simultaneously, the percentage of the eGFP-expressing cells was elevated to some extent. The vector with both MARs in forward orientation flanking the expression cassette yielded the highest transgene expression levels (2.5-fold). The orientation (forward or reverse) of the MARs did not present a significant difference when added in the same site. In addition, transgene expression levels were not exclusively dependent on transgene copy numbers. Bioinformatic analysis indicated that some specific transcription factors may contribute to the transcriptional process. In conclusion, two MARs in a forward orientation and flanking the expression cassette comprised the optimal construct for improving the stable transgene expression in the CHO-K1 cells. The effects may be related to specific transcription factors, such as PRDM1 and REL.

Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration

It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

A Stable Genetic Transformation System and Implications of the Type IV Restriction System in the Nitrogen-Fixing Plant Endosymbiont Frankia alni ACN14a.

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes that are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. The corresponding endosymbionts, Frankia and the rhizobia, however, are distantly related groups of bacteria, leading to questions about their symbiotic mechanisms and evolutionary history. To date, a stable system of electrotransformation has been lacking in Frankia despite numerous attempts by research groups worldwide. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation. Here we report the successful electrotransformation of the model strain F. alni ACN14a with an unmethylated, broad host-range replicating plasmid, expressing chloramphenicol-resistance for selection and GFP as a marker of gene expression. This system circumvented the type IV restriction barrier and allowed the stable maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the expression of egfp under the control of the nif gene cluster promoter was localized using fluorescence imaging, the expression of nitrogen fixation in nitrogen-limited culture was localized in Frankia vesicles but not in hyphae. The ability to separate gene expression patterns between Frankia hyphae and vesicles will enable deeper comparisons of molecular signaling and metabolic exchange between Frankia-actinorhizal and rhizobia-legume symbioses to be made, and may broaden potential applications in agriculture. Further downstream applications are possible, including gene knock-outs and complementation, to open up a range of experiments in Frankia and its symbioses. Additionally, in the transcriptome of F. alni ACN14a, type IV restriction enzymes were highly expressed in nitrogen-replete culture but their expression strongly decreased during symbiosis. The down-regulation of type IV restriction enzymes in symbiosis suggests that horizontal gene transfer may occur more frequently inside the nodule, with possible new implications for the evolution of Frankia.

Generation of microRNA-30e-producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect on in vitro immune responses

MicroRNAs (miRNAs) are non-coding small RNAs involved in the regulation of gene expression. In the present study, we firstly reported the use of a fish RNA virus, viral hemorrhagic septicemia virus (VHSV), as a delivery vehicle of a miRNA-30e, and the effect of miR-30e produced by the recombinant VHSV on the immune responses of Epithelioma papulosum cyprini (EPC) cells was investigated. The expression of functional miR-30e using a CMV promoter-driven vector was verified by the significantly lower eGFP expression in cells transfected with a vector containing miR-30e sponge sequence than that in cells transfected with a control vector that had mutated miR-30e sponge sequence. Furthermore, the down-regulation of reporter gene containing 3′-UTR of NF-κb inhibitor α-like protein B (NFκbiαb) by miR-30e was demonstrated, suggesting that miR-30e overexpression can increase immune responses related to NF-κB activation through inhibition of IκB. A miR-30e-expressing recombinant VHSV (rVHSV-A-miR30e) that had primary microRNA-30e sequence between N and P genes was rescued using the reverse genetic method, and the successful expression of miR-30e in the cells infected with rVHSV-A-miR30e was demonstrated using Northern blot and qRT-PCR. Cells infected with rVHSV-A-miR30e showed the increase of NF-κB activation and type I interferon induced genes expression, suggesting that rVHSV-A-miR30e can produce functional miR-30e in fish cells, and VHSV can be used as a vehicle to deliver functional microRNAs in fish.

Flock House Virus RNA1 with a Long Heterologous Sequence at the 3'-end Can Replicate in Mammalian Cells and Mediate Reporter Gene Expression.

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'- end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28°C but weak at 30°C. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.

Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line.

Transgenic birds are commonly used for time-lapse imaging and fate mapping studies in developmental biology. When researchers use transgenic birds expressing fluorescent protein, they need to understand the integration site of the transgene in the genome and the intensity of fluorescence in the tissues of interest. In this study, we determined the integration site of the transgene and fluorescence property of developing organs in our transgenic chicken line generated by lentivirus infection. The transgene was localized between exons 3 and 4 of MED27. Some homozygotes and heterozygotes appeared to be lethal at early embryonic stages. We performed histological analysis of EGFP expression in transgenic embryos at St. 14, 17, and 24 by immunohistochemistry with anti-GFP antibody on paraffin sections. Next, we cut cryosections and quantified direct EGFP intensity from the transgene in each tissue without performing immunohistochemistry. These results revealed that EGFP intensity in each tissue was unique in developing embryos and changed according to developmental stages. Finally, we demonstrated that EGFP-expressing cells in a micromass culture with co-culturing wild-type cells were clearly distinguishable via live cell imaging. These results provide essential information on the potential of our transgenic line and indicate that these transgenic chicken lines are useful for research associated with developmental biology.

A Subset of Olfactory Sensory Neurons Express Forkhead Box J1-Driven eGFP.

Forkhead box protein J1 (FOXJ1), a member of the forkhead family transcription factors, is a transcriptional regulator of motile ciliogenesis. The nasal respiratory epithelium, but not olfactory epithelium, is lined with FOXJ1-expressing multiciliated epithelial cells with motile cilia. In a transgenic mouse where an enhanced green fluorescent protein (eGFP) transgene is driven by the human FOXJ1 promoter, robust eGFP expression is observed not only in the multiciliated cells of the respiratory epithelium but in a distinctive small subset of olfactory sensory neurons in the olfactory epithelium. These eGFP-positive cells lie at the extreme apical part of the neuronal layer and are most numerous in dorsal-medial regions of olfactory epithelium. Interestingly, we observed a corresponding small number of glomeruli in the olfactory bulb wherein eGFP-labeled axons terminate, suggesting that the population of eGFP+ receptor cells expresses a limited number of olfactory receptors. Similarly, a subset of vomeronasal sensory neurons expresses eGFP and is distributed throughout the full height of the vomeronasal sensory epithelium. In keeping with this broad distribution of labeled vomeronasal receptor cells, eGFP-labeled axons terminate in many glomeruli in both anterior and posterior portions of the accessory olfactory bulb. These findings suggest that Foxj1-driven eGFP marks a specific population of olfactory and vomeronasal sensory neurons, although neither receptor cell population possess motile cilia.

The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds in vivo

ABSTRACTVascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-Axxx and anti-angiogenic, anti-permeability VEGF-Axxxb. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-Axxx) and eGFP expression denotes distal splice site selection (VEGF-Axxxb). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-Axxx and VEGF-Axxxb. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo

Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli

BackgroundAcrylic acid (AA) is a widely used commodity chemical derived from non-renewable fossil fuel sources. Alternative microbial-based production methodologies are being developed with the aim of providing “green” acrylic acid. These initiatives will benefit from component sensing tools that facilitate rapid and easy detection of in vivo AA production.ResultsWe developed a novel transcriptional sensor facilitating in vivo detection of acrylic acid (AA). RNAseq analysis of Escherichia coli exposed to sub-lethal doses of acrylic acid identified a selectively responsive promoter (PyhcN) that was cloned upstream of the eGFP gene. In the presence of AA, eGFP expression in E. coli cells harbouring the sensing construct was readily observable by fluorescence read-out. Low concentrations of AA (500 μM) could be detected whilst the closely related lactic and 3-hydroxy propionic acids failed to activate the sensor. We further used the developed AA-biosensor for in vivo FACS-based screening and identification of amidase mutants with improved catalytic properties for deamination of acrylamide to acrylic acid.ConclusionsThe transcriptional AA sensor developed in this study will benefit strain, enzyme and pathway engineering initiatives targeting the efficient formation of bio-acrylic acid.

Single-cell kinetics of siRNA-mediated mRNA degradation

RNA interference (RNAi) enables the therapeutic use of small interfering RNAs (siRNAs) to silence disease-related genes. The efficiency of silencing is commonly assessed by measuring expression levels of the target protein at a given time point post-transfection. Here, we determine the siRNA-induced fold change in mRNA degradation kinetics from single-cell fluorescence time-courses obtained using live-cell imaging on single-cell arrays (LISCA). After simultaneous transfection of mRNAs encoding eGFP (target) and CayRFP (reference), the eGFP expression is silenced by siRNA. The single-cell time-courses are fitted using a mathematical model of gene expression. Analysis yields best estimates of related kinetic rate constants, including mRNA degradation constants. We determine the siRNA-induced changes in kinetic rates and their correlations between target and reference protein expression. Assessment of mRNA degradation constants using single-cell time-lapse imaging is fast (<30 h) and returns an accurate, time-independent measure of siRNA-induced silencing, thus allowing the exact evaluation of siRNA therapeutics.

A new transgenic reporter line reveals Wnt-dependent Snai2 re-expression and cranial neural crest differentiation in Xenopus

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snai2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snai2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fluorescent protein (eGFP) driven by the snai2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the differentiating CNC. This transgenic line can be used directly to detect deficiencies in CNC development at various stages, including subtle perturbation of CNC differentiation. In situ hybridization and immunohistochemistry confirm that Snai2 is re-expressed in the differentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the differentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snai2 expression and impaired differentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling is required for snai2 re-expression and CNC differentiation.

Transcriptional Enhancers in the FOXP2 Locus Underwent Accelerated Evolution in the Human Lineage.

Unique human features, such as complex language, are the result of molecular evolutionary changes that modified developmental programs of our brain. The human-specific evolution of the forkhead box P2 (FOXP2) gene-coding region has been linked to the emergence of speech and language in the human kind. However, little is known about how the expression of FOXP2 is regulated and whether its regulatory machinery evolved in a lineage-specific manner in humans. In order to identify FOXP2 regulatory regions containing human-specific changes, we used databases of human-accelerated noncoding sequences or HARs. We found that the topologically associating domain determined using developing human cerebral cortex containing the FOXP2 locus includes two clusters of 12 HARs, placing the locus occupied by FOXP2 among the top regions showing fast acceleration rates in noncoding regions in the human genome. Using in vivo enhancer assays in zebrafish, we found that at least five FOXP2-HARs behave as transcriptional enhancers throughout different developmental stages. In addition, we found that at least two FOXP2-HARs direct the expression of the reporter gene EGFP to foxP2-expressing regions and cells. Moreover, we uncovered two FOXP2-HARs showing reporter expression gain of function in the nervous system when compared with the chimpanzee ortholog sequences. Our results indicate that regulatory sequences in the FOXP2 locus underwent a human-specific evolutionary process suggesting that the transcriptional machinery controlling this gene could have also evolved differentially in the human lineage.

AAV GCG-EGFP, a new tool to identify glucagon-secreting \u03b1-cells

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.