EGFP Expression Research Articles

Systematic Comparison of Plant Promoters in Nicotiana spp. Expression Systems.

We report a systematic comparison of 19 plant promoters and 20 promoter-terminator combinations in two expression systems: agroinfiltration in Nicotiana benthamiana leaves, and Nicotiana tabacum BY-2 plant cell packs. The set of promoters tested comprised those not present in previously published work, including several computationally predicted synthetic promoters validated here for the first time. The expression of EGFP driven by different promoters varied by more than two orders of magnitude and was largely consistent between two tested Nicotiana systems. We confirmed previous reports of significant modulation of expression by terminators, as well as synergistic effects of promoters and terminators. Additionally, we observed non-linear effects of gene dosage on expression level. The dataset presented here can inform the design of genetic constructs for plant engineering and transient expression assays.

Dysfunctional Clusterin-Positive Hematopoietic Stem Cells Expand with Aging

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes dysregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old), and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq data revealed that transcriptomic profiles of HSCs change with aging, although middle-aged and aged HSCs showed very similar profiles to each other. scRNA-seq data divided young and aged HSCs into 2 and 3 clusters, respectively. While both young and aged HSCs had clusters with myeloid or lymphoid gene signature, the cluster with myeloid signature dominated in aged HSCs, in which aged gene signature (Nupr1, Mt1, Mt2, Selp) was highly enriched. Clusterin (Clu) was also significantly up-regulated in this cluster. Clu encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis (Itakura et al., JCB 2020). We hypothesized that Clu would be useful as a marker of aged HSCs, and conducted further analysis using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory elements of the Clu gene. Bone marrow (BM) analysis of Clu-reporter mice revealed that Clu expression is confined to a part of HSCs in BM and significantly up-regulated within the HSC compartment with aging (10 and 70 % of HSCs are Clu-positive in young and middle-aged mice, respectively). In competitive repopulation assays, Clu-positive HSCs showed severely impaired repopulation capacity and preferentially differentiated into myeloid cells while Clu-negative HSCs largely retained repopulation capacity and showed balanced differentiation. We analyzed the transcriptomic profile of Clu-positive HSCs with aging. RNA-sequence analysis revealed that young and middle-aged Clu-positive HSCs were significantly enriched for aged HSC gene signature and myeloid-biased HSC signature compared to Clu-negative HSCs. We evaluated the transcriptional regulation mechanism of Clu by Assay for Transposase-Accessible Chromatin sequence (ATAC-seq) analysis. Chromatin accessibility at the Clu promoter was already open in young HSCs, and largely enhanced in Clu-positive HSCs but not in Clu-negative HSCs among middle-aged HSCs. The open chromatin region at the Clu promoter had binding motifs of AP-1 and STAT family transcription factors, suggesting that external signals or stresses activate Clu expression. These results suggest that Clu-positive cells represent myeloid-biased HSCs which expand with aging and that Clu serves as a faithful marker to trace alterations in HSC heterogeneity with aging.

Fluorescent Tagging of Endogenous Beta-Globin Gene in S-HUDEP2 Cell Model for Live Cell Monitoring of Beta-Globin Expression and Regulation

Introduction: The translation of β-globin (HBB) gene is crucial for adult hemoglobin (HbA) formation, and naturally occurring mutations in HBB cause disorders of erythropoiesis. Therefore, understanding the mechanisms governing HBB regulation and expression has been an active area of research. We aim to develop a cell model to monitor HBB expression in live cells as a tool to gain better understanding of HBB expression, regulation and for the discovery of novel therapeutic targets. Methods: Sickle Human Umbilical Cord Derived Erythroid Progenitor cells (S-HUDEP2) are immortalized CD34+ hematopoietic stem and progenitor cells with homozygous sickle mutation that exclusively produce sickle hemoglobin upon erythroid differentiation. To generate the cell model in S-HUDEP2, we delivered HiFi SpCas9/gRNA targeting the C-terminus of HBB together with a double-stranded DNA donor template encoding self-cleaving peptide, eGFP, and β-globin polyA signal flanking homology arms for simultaneous expression of β-globin and eGFP under the HBB promoter. We created this clonal cell model and confirmed biallelic precise in-frame targeted integration by PCR, Sanger sequencing and digital droplet PCR copy number assay to ensure simultaneous monitoring of both HBB alleles. Results: To validate this model, we delivered HiFi SpCas9/R-66S sgRNA as ribonucleoprotein (RNP) targeting HBB currently in pre-clinical studies for sickle cell anemia (SCA). With RNP delivery, CRISPR/Cas9 double-strand breaks (DSBs) will lead to insertions and deletions (indels); we hypothesized that varying indel frequencies and patterns will result in change in GFP expression. After RNP delivery, we conducted 7 days of in vitro cellular expansion to allow for completion of editing and stabilization of GFP signal followed by 5 days of in vitro cellular differentiation. We identified 3 distinct populations based on GFP expression: GFP-, GFPlow, and GFPhigh. These cells were bulk sorted, genomic DNA was extracted and we analyzed indel patterns for each population through targeted amplicon Next Generation Sequencing (NGS). Each population had a unique indel pattern: confirming that GFP expression is based on genotype. In addition, an increase in GFP mean fluorescent intensity (MFI) was seen after in vitro differentiation with preservation of these distinct groups; indicative of expected increase in HBB transcription. Based on these observations, through use of ddPCR copy number assay, we determined that the creation of CRISPR/Cas9 mediated large deletion (>200bp) is the underlying mechanism leading to GFP loss seen in GFP- andGFPlow populations. Nonsense mutations occurring early in exon 1 of HBB resulting in premature translation-termination codons are resistant to nonsense mediated mRNA decay (NMD), a well-known cellular surveillance mechanism. We hypothesized that RNP induced small frameshift indels in exon 1 allow for NMD escape thus preserving HBB translation and GFP expression while large deletions do not, leading to GFP loss. To further validate this model and define its potential for broad applicability, we confirmed its ability to undergo normal differentiation based on erythroid specific cell markers when compared to untagged S-HUDEP2. We also confirmed our model's capacity to form Hb tetramer with α-chains using Hb native PAGE which allows for identification of intact Hb tetramers based on reference migration. Conclusion: We have successfully established and validated a clonal cell model with GFP tagged β-globin, which can be used to monitor HBB expression in live cells via GFP expression, eliminating the need for cellular fixation and HbA antibody staining. Our model may provide a useful tool for a broad range of applications, including the examination of cellular mechanisms underlying HBB expression and the screening of potential therapeutic targets for hemoglobinopathies and other disorders of erythropoiesis.

Study of Pathogenesis Using Fluorescent Strain of Cordyceps farinosa Revealed Infection of Thitarodes armoricanus Larvae via Digestive Tract.

Cordyceps farinosa is often utilized as a biocontrol agent because of its wide host range, strong lethality, and safety for mammals. Artificial rearing of Thitarodes armoricanus larvae is a prerequisite for the artificial cultivation of Chinese cordyceps, and C. farinosa is the most lethal pathogenic fungus during the rearing process. However, the infection process of C. farinosa is still unclear. In this study, we cloned the promoter of the C. farinosa glyceraldehyde 3-phosphate dehydrogenase gene, constructed the EGFP expression cassette, and integrated it into the C. farinosa genome via Agrobacterium transformation. We obtained a fluorescent strain for better observation of the infection process. Using two different inoculation methods of the fluorescent strain, we observed the traditional infection process through the body surface as well as through the digestive tract via feeding. Both infection modes can lead to larval death and mummification. Our findings demonstrated that during the artificial rearing of T. armoricanus, preventing C. farinosa pollution should be an important part of the disinfection of the rearing environment.

Mycobacterium leprae hsp18 promoter-EGFP transcriptional fusion construct: Environmental stress and strain-specific expression

The Hsp18 protein is a major T-cell antigen of Mycobacterium leprae belonging to the family of small heat-shock proteins. The protein is specifically regulated at post-translational level during the intracellular growth of M. leprae within macrophages due to auto-phosphorylation, indicating its importance in the survival of the bacterium. The promoter and regulatory sequences that control hsp18 expression are located within a 256-bp sequence upstream of the translation start site. However, there are no studies describing either characterization of the hsp18 promoter or its genetic regulation. Therefore, we constructed an hsp18-EGFP transcriptional fusion in an E. coli–Mycobacterium shuttle vector. A 168-bp sequence comprising the hsp18 promoter was cloned upstream of the EGFP gene and transformed in M. smegmatis, and the integration of the construct was confirmed by Southern hybridization. hsp18 promoter activity was measured by analyzing EGFP expression in M. smegmatis and Escherichia coli grown under different environmental stress conditions normally encountered by M. leprae in vivo. We found that the 168-bp upstream sequence of hsp18 could function as a promoter, and the regulation of hsp18 expression was host-, environmental stress-, and temperature-dependent. Appreciable EGFP expression was detected in M. smegmatis grown under normal conditions, and theexpression was significantly increased by environmental stress. However, EGFP expression was observed in E. coli only under stress conditions. Comparative sequence analysis revealed the putative sigma factor C (SigC)-binding site within the 168-bp promoter sequence of hsp18, which might be involved in the regulation of hsp18 expression during stress conditions in M. leprae. Thus, our data demonstrated the transcriptional regulation of hsp18 expression in response to different environmental stress conditions, possibly through SigC in Mycobacterium. Further, this shuttle vector could be used for the functional characterization of M. leprae genes in heterologous systems.

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke.

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies.

A History of Mechanical Tension Depletes Lgr6+ Epidermal Stem Cells

PURPOSE: Recent research found the epidermal Lgr6 stem cell population to be progressively depleted under mechanical tension, with a paralleled increase in the Lgr6 descendant population.1 This study aims to determine if there is a reversal of tension-induced skin growth characteristics, specifically if the Lgr6 stem cell population recovers after tension is removed. Our results will guide future research in therapeutic interventions for optimal skin recovery following tension, such as seen with bariatric surgery and the resulting excess skin from rapid weight loss. METHOD: Genetic Lgr6-EGFP-Cre-ERT2;tdTomato mice (EGFP=Lgr6, tdT=Lgr6 descendants) underwent controlled expansion (E) of the back skin. A tissue expander was surgically placed under the back skin on day zero, rested for 7 days, expanded over 10 days with a total of 24 mL saline, rested for 14 days, then deflated of saline at post-injection day 14 (PI-14) or deflation day zero (DF-0). Skin over the expander was resected, embedded in paraffin, and sectioned at 4µm. Cell populations were defined using immunofluorescence and quantified by relative expression and cell counting (ImageJ). Control mice underwent expander surgery but did not undergo saline expansion (non-expanded, NE). NE and E mice (n=3 each) were compared for PI-14, DF-14, and DF-56. We used two-tailed t-test with alpha set at 0.05. RESULTS: Relative expression of EGFP decreased from NE to both DF-14 (p=0.0474) and DF-56 (p=0.0477), while relative expression of tdT increased from NE to DF-56 (p=0.0125). EGFP+ cells decreased from NE to PI-14, DF-14, and DF-56 (p=0.0114 for all), while tdT+ cells increased from NE to PI-14, DF-14, and DF-56 (p=0.0086, p=0.0133, p=0.00002). Proliferating cells (Ki67+) increased from NE to PI-14, DF-14, and DF-56 (p=0.00007, p=0.00001, p=0.0417), but decreased from DF-14 to DF-56 (p=0.0278). Of the proliferating cell population, the [Ki67+tdT+] population increased from NE to DF-56 (p=0.0002). Cytokeratin 5 (K5) relative expression increased from NE to PI-14, DF-14, and DF-56 (p=0.0109, p<0.00001, p<0.00001). The number of epidermal keratinocyte layers increased from NE to PI-14, DF-14, and DF-56 (p<0.00001, p<0.00001, p=0.0043), with a decrease from DF-14 to DF-56 (p=0.0020). CONCLUSION: The epidermal Lgr6 stem cell population remained severely depleted following discontinuation of mechanical tension. Conversely, both the Lgr6 descendent population and K5 stem cell expression continued to increase while under tension and following release of tension. A prolonged history of discontinued tension revealed that the majority of the proliferating cell population was from Lgr6 progeny. Together, these observations suggest that in skin with a history of tension: (1) the Lgr6 population is permanently depleted, (2) the majority of the epidermis will be from Lgr6 progeny, and (3) Lgr6 progeny and K5 stem cells will be preferentially and progressively activated over Lgr6 stem cells to maintain the epidermis. Overall, these results imply that specific stimuli will prioritize responses from specific stem cell populations, and possibly to the detriment of an existing stem cell population. REFERENCES: 1. Xue Y, Lyu C, Taylor A. Mechanical Tension Mobilizes Lgr6+ Epidermal Stem Cells to Drive Skin Growth. [provisionally accepted ahead of print February 23, 2022]. Sci Adv.

Cytoplasmic Microinjection of &lt;i&gt;piggyBac&lt;/i&gt; Transposase mRNA and Transposon Vectors for Efficient &lt;i&gt;In Vitro&lt;/i&gt; Production of Transgenic Porcine Parthenotes

The efficient production of transgenic (Tg) piglets has remained a challenge in the field of domestic animal studies. Unlike mice, the pronuclei of pig zygotes cannot be easily studied because of the abundance of lipid droplets. Therefore, the zygotes must be briefly centrifuged before pronuclear injection (PNI) to move the lipid droplets to the periphery of the zygote for PNI-mediated production of Tg piglets. However, this procedure is temporal because lipid droplets return to the original space during PNI, hampering the consecutive PNI. Cytoplasmic injection (CPI) of nucleic acids is comparatively simple than PNI because CPI does not require such pre-centrifugation. Unfortunately, CPI using purified DNA fragments is inadequate for creating Tg piglets because it is challenging to integrate nucleic acids into the host genome. &lt;em&gt;PiggyBac&lt;/em&gt; (PB), one of the transposons, is a valuable tool enabling efficient chromosomal integration of a transgene. The PB-mediated gene transfer requires two components, namely, transposase and transposons harboring the gene of interest (GOI) flanked by PB acceptor sites. We speculate that the CPI of transposase mRNA and transposons could accelerate the chromosomal integration of the GOI in pig zygotes. To prove this hypothesis, we performed CPI using transposase mRNA (super PB transposase mRNA) + transposon DNA carrying the enhanced green fluorescent protein (&lt;em&gt;EGFP&lt;/em&gt;) cDNA (referred to as “pT-EGFP”), transposase expression plasmid DNA (referred to as “pTrans”) + pT-EGFP, pT-EGFP alone, or non-transposon EGFP expression plasmid DNA using porcine parthenotes. Consequently, 50% (2/4 tested) of green-fluorescent embryos exhibited chromosomal integration of GOI. In contrast, green-fluorescent embryos derived from CPI with pTrans + pT-EGFP or pT-EGFP alone did not show chromosomal integration. We used mRNA for super PB transposase, which is an engineered hyperactive version of the wild-type PB transposase. To conclude, the PB system, based on the CPI of super PB transposase mRNA + transposon DNA, could be useful for producing Tg porcine parthenotes.

Generation of a TPH2-EGFP reporter cell line for purification and monitoring of human serotonin neurons in vitro and in vivo

SummaryGeneration of serotonin neurons (SNs) from human pluripotent stem cells (hPSCs) provides a promising platform to explore the mechanisms of serotonin-associated neuropsychiatric disorders. However, neural differentiation always yields heterogeneous cell populations, making it difficult to identify and purify SNs in vitro or track them in vivo following transplantation. Herein, we generated a TPH2-EGFP reporter hPSC line with insertion of EGFP into the endogenous tryptophan hydroxylase 2 (TPH2) locus using CRISPR-Cas9-mediated gene editing technology. This TPH2-reporter, which faithfully indicated TPH2 expression during differentiation, enabled us to obtain purified SNs for subsequent transcriptional analysis and study of pharmacological responses to antidepressants. In addition, the reporter system showed strong EGFP expression to indicate SNs, which enabled us to explore in vitro and ex vivo electrophysiological properties of SNs. In conclusion, this TPH2-EGFP reporter cell line might be of great significance for studies on human SN-related development and differentiation, drug screening, disease modeling, and cell replacement therapies.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells.

Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (q mAb ) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a q mAb of 9.1–11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.

Ectosomes and exosomes modulate neuronal spontaneous activity

Extracellular vesicles (EVs) are important mediators in intercellular communication. However, understanding the biological origin and functional effects of EVs subtypes has been challenging due to the moderate differences in their physical properties and absence of reliable markers. Here, we characterize the proteomes of ectosomes and exosomes using an improved differential ultracentrifugation protocol and quantitative proteomics. Our analyses revealed singular proteomic profiles for ectosomes and exosomes that enabled us to establish specific protein markers that can be used for their biochemical distinction. Cytoskeleton and glycolytic proteins are distinctively present in ectosomes, while endosomal sorting complexes proteins and tetraspanins are enriched in exosomes. Furthermore, annexin-A2 was identified as a specific marker for ectosomes derived from cell media and human cerebrospinal fluid. Expression of EGFP as a cytosolic reporter leads to its incorporation in EVs and enables their imaging with higher resolution. Assessment of neuronal network activity using multi-electrode array recordings demonstrated that spontaneous neuronal activity can be modulated by EVs. Ectosomes and exosomes internalization in neuronal cells disrupted their regular synchronized bursting activity, resulting in overall lower and more disorganized spiking activity. Our findings suggest that EVs cargoes reflect core intracellular processes, and their functional properties might regulate basic biological and pathological processes. SignificanceThis article presents novel approaches for studying the origin, composition, and biological effects in neuronal activity of ectosomes and exosomes. Our findings suggest that EVs cargoes reflect core intracellular processes, and their functional properties might regulate basic biological and pathological processes. Ultimately, our study also forms the foundation for future biomarker studies and for the understanding of the molecular basis of different diseases.

Simultaneous imaging of redox states in dystrophic neurites and microglia at Aβ plaques indicate lysosome accumulation not microglia correlate with increased oxidative stress

The inter-relationship between microglia dynamics and oxidative stress (Ox-stress) in dystrophic neurites (DNs) at Alzheimer’s Disease (AD) plaques may contribute to the pathological changes in neurons. We developed new in vivo imaging strategies to combine EGFP expression in microglia with neuronal expression of genetically encoded ratiometric redox sensors (rogRFP2 or roGFP1), and immunohistochemistry to investigate how microglia influence Ox-stress at amyloid plaques in 5xFAD AD mice. By simultaneously imaging microglia morphology and neuronal Ox-stress over time in vivo and in fixed brains we found that microglia preferentially enwrapped DNs exhibiting the greatest degree of Ox-stress. After microglia were partially depleted with the CSF1 receptor antagonist PLX3397, Ox-stress in DNs increased in a manner that was inversely correlated to the extent of coverage of the adjacent Aβ plaques by the remaining microglia. These data suggest that microglia do not create Ox-stress at Aβ plaques but instead create protective barriers around Aβ plaques possibly reducing the spread of Aβ. Intracranial injection of Aβ was sufficient to induce neuronal Ox-stress suggesting it to be the initial trigger of Ox-stress generation. Although Ox-stress is increased in DNs, neuronal survival is enhanced following microglia depletion indicating complex and multifactorial roles of microglia with both neurotoxic and neuroprotective components. Increased Ox-stress of DNs was correlated with higher LAMP1 and ubiquitin immunoreactivity supporting proposed mechanistic links between lysosomal accumulation in DNs and their intrinsic generation of Ox-stress. Our results suggest protective as well as neurotoxic roles for microglia at plaques and that the generation of Ox-stress of DNs could intrinsically be generated via lysosomal disruption rather than by microglia.In Brief: Simultaneous imaging of microglia and neuronal Ox-stress revealed a double-edged role for microglia in 5xFAD mice. Plaque associated microglia were attracted to and enwrapped Aβ plaques as well as the most highly oxidized DNs. After partial depletion of microglia, DNs were larger with greater levels of Ox-stress. Despite increased Ox-stress after microglia removal neuronal survival improved. Greater Ox-stress was correlated with increased levels of LAMP1 and ubiquitin thereby linking lysosome accumulation and Ox-stress in DNs.

Co-expression of an isopropanol synthetic operon and eGFP to monitor the robustness of Cupriavidus necator during isopropanol production

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered.

Directed evolution of maltose induced promoters with expanded gradient intensity

Promoters with different sensitivity and response intensity are useful tools in gene expression regulation and metabolic engineering. Maltose induced promoter Pglvc was engineered to obtain promoters with different induced expression intensities. A promoter Pglvc mutant library was built by error-prone PCR, and screened by a growth-associated method using tetracycline resistance as an indicator. A library of promoter mutants with different sensitivity and intensity was obtained, and the maltose-induced response threshold range of promoter mutants (MT2, MT3, MT4, MT6) was extended from 0-3 g/L to 0-15 g/L. Among them, the highest induced expression intensity (MT8) was about 3.15 times higher than that of the original promoter for eGFP expression, which would be useful for its application in metabolic engineering and synthetic biology.

Packaging, Purification, and Titration of Replication-Deficient Semliki Forest Virus-Derived Particles as a Self-Amplifying mRNA Vaccine Vector

Background:Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for SFV-PD SAM preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of VRP-packaged SAM for vaccine studies.Methods:pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into HEK-293 and BHK-21 cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by RT-qPCR. Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR. Conclusion:Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.

Efficient Viral Transduction in Fetal and Adult Human Inner Ear Explants with AAV9-PHP.B Vectors.

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application.

Electroporation of SUMO-His-Cre protein triggers a specific recombinase-mediated cassette exchange in HEK 293T cells

Cre recombinase is a widely-used genetic manipulation of genomic DNA. However, the conventional transfection of the DNA vectors expressing the Cre recombinase or viral transduction method yields low transfection efficiencies or insertion mutagenesis. The present paper evaluated whether the direct protein delivery of Cre recombinase through electroporation can induce the Cre-mediated recombination in the HEK 293T cells. Here, the small ubiquitin-related modifier (SUMO) -tagged His-Cre fusion protein was expressed in a soluble pattern in the Eschrichria coli (E.coli) cells, purified using affinity chromatography, and finally electroporated into the HEK 293T cells. These cells were previously transfected with three different Cre reporter vectors. The electroporation of the HEK 293T cells revealed either the activation of EGFP expression, or a decrease in RFP expression, and a concomitant increase in EGFP expression, indicating a desired recombinase-mediated cassette exchange (RMCE) event (conversion of RFP to EGFP), and a biological activity of the purified SUMO-His-Cre protein. The fusion protein is expected to serve in the Easi-CRISPR-LoxP-mediated genome editing to generate transgenic animal models.

In-vivo egfp expression in the honeybee Apis mellifera induced by electroporation and viral expression vector.

In this study we describe egfp expression induced by two techniques: in vivo electroporation and viral transduction in several cell types of the adult honeybee brain. Non-neuronal and neuronal cell types were identified and the expression persisted at least during three days. Kenyon cells, optic lobe neurons and protocerebral lobe neurons were electroporated. Astrocyte-like glia cells, fibrous lamellar glia cells and cortex glia cells were identified. Viral transduction targeted one specific type of glia cells that could not be identified. EGFP positive cells types were rather variable after electroporation, and viral transduction resulted in more homogenous groups of positive cells. We propose that these techniques remain a good alternative to transgenic animals because they potentially target only somatic cells.

A Structural Atlas of the Developing Zebrafish Telencephalon Based on Spatially-Restricted Transgene Expression.

Zebrafish telencephalon acquires an everted morphology by a two-step process that occurs from 1 to 5 days post-fertilization (dpf). Little is known about how this process affects the positioning of discrete telencephalic cell populations, hindering our understanding of how eversion impacts telencephalic structural organization. In this study, we characterize the neurochemistry, cycle state and morphology of an EGFP positive (+) cell population in the telencephalon of Et(gata2:EGFP)bi105 transgenic fish during eversion and up to 20dpf. We map the transgene insertion to the early-growth-response-gene-3 (egr3) locus and show that EGFP expression recapitulates endogenous egr3 expression throughout much of the pallial telencephalon. Using the gata2:EGFPbi105 transgene, in combination with other well-characterized transgenes and structural markers, we track the development of various cell populations in the zebrafish telencephalon as it undergoes the morphological changes underlying eversion. These datasets were registered to reference brains to form an atlas of telencephalic development at key stages of the eversion process (1dpf, 2dpf, and 5dpf) and compared to expression in adulthood. Finally, we registered gata2:EGFPbi105 expression to the Zebrafish Brain Browser 6dpf reference brain (ZBB, see Marquart et al., 2015, 2017; Tabor et al., 2019), to allow comparison of this expression pattern with anatomical data already in ZBB.

Stabilizing and Anti-Repressor Elements Effectively Increases Transgene Expression in Transfected CHO Cells.

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.