Fluorescent Protein Gene Research Articles

Functional characterization of a Δ6 fatty acid desaturase gene and its 5′-upstream region cloned from the arachidonic acidrich microalga Myrmecia incisa Reisigl (Chlorophyta)

It is suggested that Δ6 fatty acid desaturase (FAD) plays a critical role in the biosynthesis of polyunsaturated fatty acids in plants and microalgae. But why does it adapt to the changed environments such as nitrogen starvation is seldom understood. One Δ6 FAD gene ( MiD6fad ) from an arachidonic acidrich microalga Myrmecia incisa Reisigl (Chlorophyta) was first heterologously expressed in Saccharomyces cerevisiae for the identification of function. The fatty acid profile of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6FAD could convert linoleic and α - linolenic acids to γ-linolenic and stearidonic acids, respectively, demonstrating that MiD6fad encoded a Δ6 FAD. A 1 965-bp fragment of the cloned 2 347-bp 5′-upstream region of MiD6fad was next subcloned and fused upstream with green fluorescent protein (GFP) gene to replace the GAL1 promoter of the vector pYES2. The generated construct was transformed into S. cerevisiae for function determination. Confocal microscopic images of the transformed line illustrated that this inserted fragment could drive GFP expression, which was further verified by fluorescence intensity quantification and Western blot analysis using anti- GFP antibody. The conversion efficiency (approximately 2% - 3%) of MiD6FAD was much lower than the reported ω 3 FAD and Δ6 elongase in this microalga, suggesting that MiD6FAD catalysed the possible ratelimiting step for ArA biosynthesis. The presence of several putative cis -acting regulatory elements in this identified promoter sheds new light on the regulation mechanism research of Δ6 FAD transcription for the ArA production in M. incisa in changing environmental factors.

Grain width 2 (GW2) and its interacting proteins regulate seed development in rice (Oryza sativa L.)

BackgroundSeed size has been extensively studied in crop plants, as it determines crop yield. However, the mechanism of seed development remains elusive. In this study, we explored the mechanism of seed development in rice (Oryza sativa L.), and identified proteins affecting seed size.ResultsProteomic analysis showed that glyceraldehyde 3-phosphate dehydrogenase, chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) accumulated to high levels in the seeds of the natural japonica rice mutant Oochikara, which carries a loss-of-function mutation in the grain width 2 (GW2) gene; GW2 encodes a RING-type E3 ubiquitin ligase. In vitro pull-down and ubiquitination assays showed that CHT14 and PGK directly interacted with GW2 but were not ubiquitinated by GW2. Immunoblot analysis revealed that protein disulfide isomerase-like 1-1 accumulated to high levels in young developing seeds of the gw2 mutant compared with the wild type. Histochemical β-glucuronidase staining showed strong expression of GW2 in leaf and root tissues but weak expression in leaf sheaths and internodes. In addition, transformation of the green fluorescent protein (GFP) gene under the control of the GW2 promoter in rice revealed GFP expression in the aleurone layer of seeds.ConclusionsCollectively, these results suggest that GW2 regulates seed size through direct interactions with proteins involved in carbohydrate metabolism by modulating their activity or stability and controlling disulfide bond formation in various proteins during seed development. Additionally, GW2 participates in vegetative as well as reproductive growth, and protects the seed from pathogen attack.

Development of a new tobamovirus-based viral vector for protein expression in plants.

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

Colonization of Bacillus cereus NJSZ-13, a species with nematicidal activity in Masson pine (Pinus massoniana Lamb.)

Bacillus cereus NJSZ-13, an endophytic bacterium with nematicidal activity, was isolated from stems of healthy Pinus elliottii Engelm. Colonization of P. massoniana Lamb. by endophytic B. cereus was studied using scanning electron microscopy and confocal laser scanning microscopy. After the plasmid pGFP78 containing the green fluorescent protein (GFP) gene was transformed into the NJSZ-13 strain, the NJSZ-13:gfp showed the same nematicidal activity and growth curve as the wild-type strain, and the plasmid pGFP78 was stably maintained in strain NJSZ-13 for at least 96 h of bacterial cultivation on medium without antibiotics. After inoculation into Masson pine roots, colonization of the NJSZ-13:gfp strain in plant roots and stems was visualized using confocal laser scanning and the strain was enumerated in inoculated roots and stems. These results suggest that NJSZ-13:gfp is an efficient colonizer of Masson pine and can transfer vertically from roots to stems.

Local delivery of a cancer-favoring oncolytic vaccinia virus via poly (lactic-co-glycolic acid) nanofiber for theranostic purposes

Local delivery of viruses via nanomaterials has been demonstrated in the treatment of colon cancer. A cancer-favoring oncolytic vaccinia virus (CVV), which is an evolutionary cancer-favoring engineered vaccinia virus carrying the green fluorescent protein (GFP) gene, has antitumor efficacy in drug-resistant colon cancer and metastatic liver cells. Here, we present the antitumor efficacy of a CVV coated a poly lactic-co-glycolic acid (PLGA) nanofiber for local drug delivery in theranostic approaches. In vitro- and in vivo-based analyses using mouse colon carcinoma models confirmed the therapeutic efficacy of CVV-PLGA nanofibers through diagnostic localization of the tumors and reduced tumor burden as a result of enhanced apoptosis. We propose that the CVV-PLGA nanofiber may be used for theranostic purposes in cancer therapy.

Transient expression of the enhanced green fluorescent protein (egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S. horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S. horneri with analysis of molecular markers. The S. horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that GFP is an effective transgene reporter for S. horneri and that particle bombardment is a suitable method for transformation of S. horneri. Through selection of four different promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S. horneri.

Changes to the core and flanking sequences of G-box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters.

Summary Cis‐regulatory elements in promoters are major determinants of binding specificity of transcription factors (TFs) for transcriptional regulation. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of both classical (CACGTG) and variant G‐box core sequences along with different flanking sequences derived from the promoters of three different highly expressing soybean genes, were constructed and used to regulate a green fluorescent protein (gfp) gene. Use of the classical 6‐bp G‐box provided information on the base level of GFP expression while modifications to the 2–4 flanking bases on either side of the G‐box influenced the intensity of gene expression in both transiently transformed lima bean cotyledons and stably transformed soybean hairy roots. The proximal 2‐bp sequences on either flank of the G‐box significantly affected G‐box activity, while the distal 2‐bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Manipulation of the upstream 2‐ to 4‐bp flanking sequence of a G‐box variant (GACGTG), found in the proximal region of a relatively weak soybean glycinin promoter, significantly enhanced promoter activity using both transient and stable expression assays, if the G‐box variant was first converted into a classical G‐box (CACGTG). In addition to increasing our understanding of regulatory element composition and structure, this study shows that minimal targeted changes in native promoter sequences can lead to enhanced gene expression, and suggests that genome editing of the promoter region can result in useful and predictable changes in native gene expression.

Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons.

Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.

A Functional Comparison of Treatment of Intrinsic Sphincter Deficiency with Muscle-Derived and Adipose Tissue-Derived Stem Cells.

This study investigated the effect of muscle-derived stem cells (MDSCs) and adipose tissue-derived stem cells (ADSCs) in the treatment of stress urinary incontinence (SUI) and their differences in a rat model. MDSCs and ADSC were isolated from rats (n = 10), examined for their properties, and labeled with enhanced green fluorescent protein (EGFP) and β-galactosidase (β-gal) gene. Rats received bladder-neck and transurethral sphincter injection of EGFP-labeled MDSCs and β-gal gene-labeled ADSC and injection of D-Hanks as a control (n = 24 each group). At 0, 15, 30, and 60 days after cells injection, urinary voiding function was assessed by urine dynamics detector. The rats were killed to harvest their urethras for tracking of MDSCs and ADSC. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) was performed to detect smooth muscle contents. Urodynamic test showed that MDSCs and ADSC improved the function of urination in rats with intrinsic sphincter deficiency (ISD), and effect of MDSCs-treatment was more pronounced. In addition, histologic analysis showed that the MDSCs and ADSC-treated groups had significantly higher myosin and α-smooth muscle actin (α-SMA) content than the control group. Compared with ADSC-treated groups, the MDSCs-treated groups in myosin and α-SMA content showed the tendency of increase. In summary, MDSCs and ADSCs have obvious effects in the treatment and/or prevention of ISD and transplantation of MDSCs is more effective than ADSC. © 2018 IUBMB Life, 70(10):976-984, 2018.

Generation of transgenic-cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein.

Huanjiang Xiang pig is a unique native minipig breed originating in Guangxi, China, and has great utility value in agriculture and biomedicine. Reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) and SCNT-mediated genetic modification show great potential value in genetic preservation and utilization of Huanjiang Xiang pigs. Our previous work has successfully produced cloned and transgenic-cloned embryos using somatic cells from a Huanjiang Xiang pig. In this study, we firstly report the generation of transgenic-cloned Huanjiang Xiang pigs carrying an enhanced green fluorescent protein (eGFP) gene. A total of 504 SCNT-derived embryos were transferred to two surrogate recipients, one of which became pregnant and gave birth to three live piglets. Exogenous eGFP transgene had integrated in all of the three Huanjiang Xiang piglets identified by genotyping. Furthermore, expression of eGFP was also detected from in vitro cultured skin fibroblast cells and various organs or tissues from positive transgenic-cloned Huanjiang Xiang pigs. The present work provides a practical method to preserve this unique genetic resource and also lays a foundation for genetic modification of Huanjiang Xiang pigs with improved values in agriculture and biomedicine.

Construction of lung tumor cells using RFP and Fluc dual-labeled system

Objective To establish a human lung cancer cell line that can stably express firefly luciferase (Fluc) and red fluorescent protein (RFP) gene so as to lay the foundation for the further establishment of a live-imaging lung cancer xenograft model in nude mice and therapeutic research. Methods The lentiviral vector pHBLV-Fluc-RFP containing luciferase and red fluorescent protein was constructed and then transfected into 293T cells for virus packaging. The complete virus was used to infect human lung cancer cell lines A549, H1975 and human B-cell lymphoma cell line K562. The stable cell lines were obtained by puromycin selection. Fluorescence microscopy and quantitative PCR were used to confirm the RFP and Fluc expression. Results The lentiviral vector pHBLV-Fluc-RFP was successfully constructed. Cancer cell line A549, H1975 and K562 stably expressing Fluc and RFP was obtained. The real-time quantitative PCR results showed that the relative expression of Fluc gene in the three stable infected cells was much higher than that in the corresponding wild-type cells, and the differences were statistically significant(all P<0.05). Conclusion The human lung cancer cell line A549, H1975 and human B-cell lymphoma cell line K562 with dual expression of RFP and Fluc were obtained, which provided a new model of fluorescent cells for in vivo imaging of immunodeficient mouse models such as nude mice. Key words: Red fluorescent protein; Firefly luciferase; Lentivirus; Lung cancer cell fluorescence model

Tracer technology of probiotics based on optical imaging

Probiotics refers to live microorganisms, which is beneficial for host health mainly by adjusting the gastrointestinal tract flora, improving intestinal epithelial barrier function and regulating immune function. Common probiotic strains include Bifidobacteria (such as Bifidobacterium longum, Bifidobacterium thermophilus ), Lactobacillus (such as Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri ), Bacillus (such as Bacillus subtilis ), yeast Bacteria (such as saccharomyces ) etc. In the past, probiotics were counted by analyzing the number of bacteria in feces or dissecting animals gastrointestinal tracts at different times. The action mechanism of probiotics is still unclear. Genetic labeling of probiotics by modern biological techniques is one of important research methods and it provides a basis for researching the mechanisms of probiotics. Tracer technology of probiotics is based on optical imaging, which includes bioluminescence, traditional fluorescence (fluorescent protein and fluorescent dye) and nanomaterials fluorescence. The main applications in the past decade are bioluminescence technology and fluorescence technology. The difference between them is that bioluminescence refers to self-luminescence. It catalyzes the substrate reaction and emits visible light by expressing and secreting different sources of luciferase. This reaction does not require exciting light. The luciferase derived from bacteria does not require the addition of substrate through expressing Lux CDABE. The luminescent reaction catalyzed by luciferase derived from fungus has a longer luminescence wavelength and is more permeable to tissue for imaging, but this reaction requires the substrate. Fluorescence is the expression of different fluorescent proteins, which emit fluorescence under the stimulation of exciting light. This reaction does not require the participation of the substrate, but requires external exciting light, so the background interference is relatively intense. Nanomaterial luminescence is a new type of probiotic tracer technology in recent years. The fluorescence imaging of new nanomaterials mainly includes quantum dots, upconversion nanomaterials and persistent luminescence nanomaterials. Persistent luminescence nanomaterials can even maintain the luminescence phenomenon for a few days after stopping the excitation. Compared to traditional fluorescence imaging technology, fluorescent imaging of nanomaterials may have more extensive applications, due to the lack of introduction of fluorescent protein genes. However fluorescent imaging of nanomaterials still has some disadvantages, such as high cost and toxicity etc. The characteristics, advantages and disadvantages of the three techniques are reviewed in order to provide theoretical and reference basis for further development and research on the tracing technology of probiotics.

Establishment of Transgenesis in the Demosponge Suberites domuncula.

Sponges (Porifera) represent one of the most basally branching animal clades with key relevance for evolutionary studies, stem cell biology, and development. Despite a long history of sponges as experimental model systems, however, functional molecular studies are still very difficult to perform in these animals. Here, we report the establishment of transgenic technology as a basic and versatile experimental tool for sponge research. We demonstrate that slice explants of the demosponge Suberites domuncula regenerate functional sponge tissue and can be cultured for extended periods of time, providing easy experimental access under controlled conditions. We further show that an engineered expression construct driving the enhanced green fluorescence protein (egfp) gene under control of the Suberites domuncula β-actin locus can be transfected into such tissue cultures, and that faithfully spliced transcripts are produced from such transfected DNA. Finally, by combining fluorescence-activated cell sorting (FACS) with quantitative PCR, we validate that transfected cells can be specifically reisolated from tissue based on their fluorescence. Although the number of detected enhanced green fluorescent protein (EGFP)-expressing cells is still limited, our approach represents the first successful introduction and expression of exogenous DNA in a sponge. These results represent a significant advance for the use of transgenic technology in a cornerstone phylum, for instance for the use in lineage tracing experiments.

Rainbow-Seq: Combining Cell Lineage Tracing with Single-Cell RNA Sequencing in Preimplantation Embryos

SummaryWe developed the Rainbow-seq technology to trace cell division history and reveal single-cell transcriptomes. With distinct fluorescent protein genes as lineage markers, Rainbow-seq enables each single-cell RNA sequencing (RNA-seq) experiment to simultaneously decode the lineage marker genes and read single-cell transcriptomes. We triggered lineage tracking in each blastomere at the 2-cell stage, observed microscopically inequivalent contributions of the progeny to the two embryonic poles at the blastocyst stage, and analyzed every single cell at either 4- or 8-cell stage with deep paired-end sequencing of full-length transcripts. Although lineage difference was not marked unequivocally at a single-gene level, it became clear when the transcriptome was analyzed as a whole. Moreover, several groups of novel transcript isoforms with embedded repeat sequences exhibited lineage difference, suggesting a possible link between DNA demethylation and cell fate decision. Rainbow-seq bridged a critical gap between division history and single-cell RNA-seq assays.

Herpes ICP8 protein stimulates homologous recombination in human cells.

Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red homologous recombination protein Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red Synaptase/Exonuclease pair (SynExo) in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a “humanized” version of Beta protein from phage λ. ICP8 significantly enhanced gene targeting rates in HEK 293T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.

Fluorescence detection of DNA mismatch repair in human cells

Mismatched base pairs, produced by nucleotide misincorporation by DNA polymerase, are repaired by the mismatch repair (MMR) pathway to maintain genetic integrity. We have developed a method for the fluorescence detection of the cellular MMR ability. A mismatch, which would generate a stop codon in the mRNA transcript unless it was repaired, was introduced into the gene encoding the enhanced green fluorescent protein (EGFP) in an expression plasmid. When MMR-proficient HeLa cells were transformed with this plasmid, the production of active EGFP was observed by fluorescence microscopy. It was assumed that the nick required to initiate the MMR pathway was produced non-specifically in the cells. In contrast, fluorescence was not detected for three types of MMR-deficient cells, LoVo, HCT116, and DLD-1, transformed with the same plasmid. In addition, the expression of a red fluorescent protein gene was utilized to avoid false-negative results. This simple fluorescence method may improve the detection of repair defects, as a biomarker for cancer diagnosis and therapy.

Transfer of Green Fluorescent Protein (GFP) Gene to Betta splendens Embryos by Transfection and Electroporation Methods

Transfection and electroporation method shave a high possibility to apply towards transgenic production of small eggs size fish species. This study aimed to examine the potential of transfection and electroporation methods to use for transferring a foreign gene into betta fish (Betta splendens) embryos using green fluorescent protein (GFP) gene as a model. Fish were spawned naturally in the ratio of male: female was 1:1, then a total of 200 eggs were taken for each treatment. Transfection was performed for 30 minutes (room temperature of about 25 °C) at two-cell stage of embryos using transfast reagent. Transfection reaction consisted of 0.75 µL transfast reagent, 0.25 µL GFP expression vector (DNA concentration: 50 µg/µL) and 99 µL NaCl solution (concentration: 0,95%). Electroporation was performed using 125 volt cm-1, 3 times pulse frequency at one second interval and pulse length of 7 micro seconds. A volume of 800 µL GFP expression vector solution (DNA concentration: 50 µg/ µL) in PBS was used for electroporation. The successful of foreign gene transfer was determined by PCR method with GFP specific primers. The results showed that hatching rate of eggs in transfection treatment was 67.08%, while the electroporation was 72.09%. Survival of larvae in transfection treatment was 73.00%, while the electroporation was 75.00%. The results of PCR analysis showed that transfection method allowed 65% of the survived fish carrying GFP gene, whereas the electroporation method was 70%. Thus, foreign gene transfer in betta fish can be conducted using the transfection and electroporation methods.

Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors

Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression.

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.

The microRNA expression profiles in extracellular vesicles from HeLa cancer cells in response to cationic lipid- or polyethylenimine-mediated gene delivery

Intercellular communication is known to be involved in various stages of tumour development and metastasis through the secretion of extracellular vesicles (EVs) containing messengers such as microRNAs (miRNAs). Therefore, this study explored miRNA profiles in cancer cell-derived EVs after non-viral gene delivery in order to better understand the molecular information of intercellular communication in cancer cells after gene delivery. Two commonly used non-viral vectors (Lipofectamine 2000 and jet polyethylenimine) were used for the delivery of gene fluorescent protein plasmid in HeLa cancer cells. EVs were extracted and the contents of their RNA were subjected to the next-generation sequencing. In order to illustrate the common characteristics of non-viral vectors in the cancer cells, two overlapped up-regulated miRNAs (hsa-miR-143-3p and hsa-miR-193b-3p) were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction in the secreted EVs in response to both lipoplexes and polyplexes. The prediction of target genes and molecular pathways involved in these two miRNAs were determined, and the protein expressions related to the pathways of cell death and stress in HeLa cells were identified. Hsa-miR-143-3p and hsa-miR-193b-3p were found to be up-regulated by the use of different non-viral vectors and can thus serve as potential targets of non-viral cancer gene therapy.