Green Fluorescent Protein Gene Research Articles

Long-duration leptin transgene expression in dorsal vagal complex does not alter bone parameters in female Sprague Dawley rats

The hypothalamus and dorsal vagal complex (DVC) are both important for integration of signals that regulate energy balance. Increased leptin transgene expression in either the hypothalamus or DVC of female rats was shown to decrease white adipose tissue and circulating levels of leptin and adiponectin. However, in contrast to hypothalamus, leptin transgene expression in the DVC had no effect on food intake, circulating insulin, ghrelin and glucose, nor on thermogenic energy expenditure. These findings imply different roles for hypothalamus and DVC in leptin signaling. Leptin signaling is required for normal bone accrual and turnover. Leptin transgene expression in the hypothalamus normalized the skeletal phenotype of leptin-deficient ob/ob mice but had no long-duration (≥10 weeks) effects on the skeleton of leptin-replete rats. The goal of this investigation was to determine the long-duration effects of leptin transgene expression in the DVC on the skeleton of leptin-replete rats. To accomplish this goal, we analyzed bone from three-month-old female rats that were microinjected with recombinant adeno-associated virus encoding either rat leptin (rAAV-Leptin, n = 6) or green fluorescent protein (rAAV-GFP, control, n = 5) gene. Representative bones from the appendicular (femur) and axial (3rd lumbar vertebra) skeleton were evaluated following 10 weeks of treatment. Selectively increasing leptin transgene expression in the DVC had no effect on femur cortical or cancellous bone microarchitecture. Additionally, increasing leptin transgene expression had no effect on vertebral osteoblast-lined or osteoclast-lined bone perimeter or marrow adiposity. Taken together, the findings suggest that activation of leptin receptors in the DVC has minimal specific effects on the skeleton of leptin-replete female rats.

Enabling Non-invasive Tracking of Vascular Endothelial Cells Derived from Induced Pluripotent Stem Cells Using Nuclear Imaging.

The absence of clinically applicable imaging techniques for continuous monitoring of transplanted cells poses a significant obstacle to the clinical translation of stem cell-based therapies for vascular regeneration. This study aims to optimize a clinically applicable, non-invasive imaging technique to longitudinally monitor vascular endothelial cells (ECs) for vascular regeneration in peripheral artery disease (PAD). Human induced pluripotent stem cells (HiPSCs) were employed to generate ECs (HiPSC-ECs). Lentiviral vectors encoding human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (eGFP) genes were introduced to HiPSCs and HiPSC-ECs at varying multiplicities of infection (MOI). Through a combination of fluorescence microscopy and flow cytometry, an optimized transduction technique for introducing hNIS-eGFP into HiPSC-ECs was established. Subsequently, single-photon emission computed tomography (SPECT) was utilized for imaging of the transduced cells in vitro and in vivo after transplantation into the gastrocnemius muscle of nude mice. Lentiviral transduction resulted in sustained co-expression of hNIS and eGFP in HiPSC-ECs when transduced post-endothelial differentiation. An optimal MOI of five yielded over 90% hNIS-eGFP expression efficiency without compromising cell viability. hNIS-eGFP+ HiPSC-ECs exhibited 99mTc uptake and were detectable through SPECT in vitro. Additionally, intramuscular injection of hNIS-eGFP+ HiPSC-ECs with MatrigelTM into the hindlimbs of nude mice enabled real-time SPECT/CT tracking, from which a reduction in signal exceeding 80% was observed within 7days. This study establishes an optimized cell modification and imaging protocol for tracking transplanted cells. Future efforts will focus on enhancing cell survival and integration via improved delivery systems, thereby advancing the potential of cell-based therapies for PAD.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV)

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells

The utilization of chicken oviductal epithelial cells (OECs) as a bioreactor to produce therapeutic proteins has shown promise, but the time taken to obtain transgenic offspring impedes efficient validation of protein production. To overcome this barrier, we focused on the immortalization of chicken OECs (cOECs) using retroviral vector-mediated c-MYC oncogene expression to establish an in vitro pre-validation system for chicken bioreactors. The resulting immortalized cOECs exhibited sustained proliferation, maintained a normal diploid chicken karyotype, and expressed key oviduct-specific genes (OVA, OVM, LYZ, AVD, and ESR1). Notably, hormonal administration of diethylstilbestrol (DES) or progesterone (P4) upregulated oviduct-specific genes in these cells. To enhance the utility of these immortalized cOECs as an in vitro validation system for chicken bioreactors, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was employed to knock-in (KI) an enhanced green fluorescence protein (EGFP) gene at the ovalbumin (OVA) locus. The resulting OVA EGFP KI immortalized cOECs secreted both EGFP and OVA proteins into the culture medium, with secretion enhanced under DES treatment. This successful integration of an exogenous gene into cOECs enhances their potential as a versatile in vitro validation system for chicken bioreactors. The established immortalized cOECs overcome previous challenges associated with long-term culture and maintenance, providing a reliable platform for efficient protein production validation. This study presents a comprehensive characterization of the immortalized cOECs, addressing critical limitations associated with in vivo systems and laying a foundation for the development of a streamlined and effective chicken bioreactor model.

Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling five cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

Background The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results The addition of a PolyR group to the C-terminal of EGFP carrying C-terminal 6×His-tag showed similar fluorescence expression levels compare to EGFP without addition of PolyR as well. Moreover, yeast with plasmid insertion showed decrease S. cerevisiae growth curve but still preserving the fluorescence EGFP. Conclusions The expression of the EGFP modified protein in S. cerevisiae is not affected by the addition of arginine.

Abstract 1431: Generation of transparent transgenic zebrafish strain to study cardiovascular functionin translational cardio-oncology research

Abstract Transgenic zebrafish lines have been extensive use in biomedical research and their primary applications. The zebrafish has proven to be an ideal tool for direct In-vivo observations of developmental processes. In vertebrates, including zebrafish and humans, the ability to obtain high-resolution, in-vivo imaging is limited due to the natural opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating blocking of pigmentation. Zebrafish casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. Our Laboratory is developing a novel transparent transgenic zebrafish model to address basic and translational cardio-oncology and experimental therapeutics. The fli1 promoter in zebrafish having an enhanced expression reporter gene of green fluorescent protein, GFP. This model allows for the in-vivo examination of the vascular system by using fluorescent and confocal microscopy. In this study, we developed transparent transgenic strain of Caspe/fli1:EGFP and integrating with recently developed casper/myl7/annexin-5:YFP and casper/fli1:EGFP strain through crossbreeding of casper and transgenic zebrafish. We have generated F1 generation of newly developed strains of Caspe/fli1:EGFP and casper/myl7/annexin-5:YFP and casper/fli1:EGFP. The presence of fluorescence expressions RFP, YFP and GFP, in crossbred progeny identified through the screening of larvae at 72 HPF stage. Expression of these fluorescent proteins determines the presence of the reporter genes. Positive transgenic larvae validated by confocal microscopy. The progenies of the F1 generation are heterozygous in nature and allow them to grow into adults to breed transparent transgenic strain to get F2 generation. This process will be continued to F2, F3, F4 and F5 generation until obtain homozygous progeny of newly developed strain. The development of casper/myl7/annexin-5:YFP x casper/fli1:EGFP will aid new dimensions in Cardio-oncology research for cardiac injury and Vasotoxicity induced by anticancer drug treatment in cancer patients. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Rohan Pasi, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simaran Hotwani, Devan-Anmol Manhiani, Kirk Kimmerling, Vishal Arora, Balakrishna L. Lokeshwar. Generation of transparent transgenic zebrafish strain to study cardiovascular functionin translational cardio-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1431.

Abstract 5547: Non-invasive monitoring of tumor biomechanics and implications for immunotherapy in HCC

Abstract Shear-wave elastography (SWE) is a novel non-invasive technique used to quantify tissue stiffness. This approach can be used to longitudinally monitor tumor biomechanics and holds promise for guiding informed clinical decision-making. This study aims to explore the impact of tumor stiffness, as evaluated by SWE, on the proliferative potential of endothelial, tumor, and immune cells in a preclinical model of hepatocellular carcinoma. Tumor tissue sections from a Buffalo-McA-RH7777 rat HCC tumor model, utilizing a rat hepatoma cell line stably expressing green fluorescent protein genes (fluc/GFP) were selected based on the degree of tumor stiffness assessed by SWE. Nodules were separated based on mean shear wave values (3.05 versus 2.26 m/s) and categorized as stiff or soft, respectively. A 6-color panel was used to assess the percentages of different tumor, immune, and endothelial phenotypes in different areas of the tumor including immune cells (CD45+), endothelial cells (CD34+), green fluorescent protein (GFP), Ki67 (cell proliferation), and class-III intermediate filaments - vimentin. Our findings demonstrated increased expression of vimentin on immune cells (CD45+) within the tumor margin interphase. Stiffer tumors exhibited a higher percentage of CD45 cells co-expressing vimentin compared to softer tumors (P=0.01), whereas softer tumors showed an increased prevalence of CD34+ cells concurrently expressing Ki67 (P=0.01). Same pattern persisted in the tumor margin interphase regarding the proliferation of tumor cells identified by the expression of GFP co-expressing Ki67 (P<0.01). These findings highlight a significant association between tumor mechanical properties and the proliferative activity of endothelial and tumor cells, suggesting a link between tissue stiffness and the regulatory mechanisms associated with cellular proliferation and aggressiveness in HCC. Additionally, the presence of vimentin in immune cells may suggest that stiffer tumors may require enhanced migratory and invasive properties to compensate for changes in tumor mechanics thus potentially influencing immune function. Citation Format: Andrea C. Cortes, Kiyoyuki Minamiguchi, Hannah Kostan, Maria S. Stenkamp, Simone Anfossi, Natalie Wall Fowlkes, Rony Avritcsher. Non-invasive monitoring of tumor biomechanics and implications for immunotherapy in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5547.

Comparative Evaluation of Transient Protein Expression Efficiency in Tissues across Soybean Varieties Using the Tsukuba System

Transient protein expression is a versatile tool with diverse applications and can be used in soybeans to study gene function, obtain mutants, and produce proteins for commercial use. However, soybeans are considered recalcitrant for agroinfiltration. Subsequent studies on soybeans have demonstrated a green fluorescent protein (GFP) expression in seedpods, but not in leaves, using syringe agroinfiltration. To evaluate agroinfiltration-based transient protein expression levels in plant cells, we used the transient expression vector pTKB3 harboring the GFP gene. Using Agrobacterium tumefaciens, vacuum agroinfiltration of the leaves and needle agroinfiltration of the seedlings of different soybean varieties were performed. GFP was transiently expressed in all of the samples. However, the Enrei and Williams 82 varieties presented better results than the other varieties in the leaf tissue, with results confirmed by immunoblot analysis, demonstrating that both varieties are good candidates for molecular biological studies. GFP expression in the seedlings was less extensive than that in the leaves, which may be due to the tissue characteristics, with Enrei showing the best results. Based on this observation, we conclude that the Tsukuba system is an effective tool that can be used for different tissues and soybean varieties.

Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes.

The bottleneck in drug discovery for central nervous system diseases is the absence of effective systemic drug delivery technology for delivering therapeutic drugs into the brain. Despite the advances in the technology used in drug discovery, such as Adeno-Associated Virus (AAV) vectors, the development of drugs for central nervous system diseases remains challenging. In this manuscript, we describe, for the first time, the development of a workflow to generate a novel brain-targeted drug delivery system that involves the generation of genetically engineered exosomes by first selecting various functional AAV capsid-specific peptides (collectively called CAPs) known to be involved in brain-targeted high-expression gene delivery, and then expressing the CAPs in frame with lysosome-associated membrane glycoprotein (Lamp2b) followed by expressing CAP-Lamp2b fusion protein on the surface of mesenchymal stem cell-derived exosomes, thus generating CAP-exosomes. Intravenous injection of green fluorescent protein (GFP) gene-loaded CAP-exosomes in mice transferred the GFP gene throughout the CNS as measured by monitoring brain sections for GFP expression with confocal microscopy. GFP gene transfer efficiency was at least 20-fold greater than that of the control Lamp2b-exosomes, and GFP gene transduction to mouse liver was low.

The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report.

Stable Cas9 (CRISPR-associated protein 9)-expressing cell lines have emerged as valuable tools in genetic research, enhancing the efficiency of the CRISPR/Cas9 system and streamlining gene editing procedures. These cell lines enable simultaneous editing of multiple genes and reduce the overall editing time. This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes. Human embryonic kidney 293LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch 8 (YhFF#8) cells were targeted for genetic modification. Transfection of human embryonic kidney 293LTV cells with pCDH-Cas9 plasmid DNA generated pseudo-viral particles. YhFF#8 cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas9 gene were determined by quantitative polymerase chain reaction. The study validated the fidelity of the Cas9 gene cassette sequence and its transcriptional activity. Transduced YhFF#8 cells exhibited green fluorescence, with antibiotic selection resulting in nearly 100% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#8-Cas9-GFP-PuroR cells using fluorescence microscopy. YhFF#8-Cas9-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research.

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells using GFP Based Reporter Systems.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

Potential benefits of Bacillus subtilis-mediated bioflocs in supplementary feeding on triangle sail mussels Hyriopsis cumingii: a pilot study of fluorescence-labeled floc-forming bacteria

As micro-particle aggregates, bioflocs may be potential baits for ingestion by triangle sail mussels and supplementary feeding raises more concerns. Besides, a plasmid carrying the green fluorescent protein (GFP) gene was transformed into floc-forming Bacillus subtilis (GFP-B. subtilis) to visualize its colonization during the bioflocs ingestion. To investigate the potential benefits of bioflocs on H. cumingii, Chlorella vulgaris diets were supplemented with nothing (control group, CG) or bioflocs accounting for the microalgae biomass at 25% (T1), 50% (T2), and 100% (T3) respectively. The community composition of bioflocs was analyzed and some species like phylum Proteobacteria, order Rhodobacterales and genus Paracoccus with potentials to improve water quality were found. Furthermore, supplementary bioflocs could reduce the concentrations of inorganic nitrogen in the aquacultural environment. Differences in growth traits across all groups were insignificant, but the digestion, metabolic and immune response of H. cumingii were promoted remarkably. Interestingly, some colonies of GFP-B. subtilis were detected in the intestines of H. cumingii from T1, suggesting H. cumingii could uptake bioflocs efficiently and intestinal colonization of B. subtilis might be accomplished. Besides, supplementary bioflocs with the highest ratio (T3) could improve the physiological status of organs and suppress the dominance of potential pathogens such as Aeromonas and Pseudomonas. It was assumed that B. subtilis might stimulate the healthy intestine development via intestinal colonization. Overall, B. subtilis-mediated bioflocs is beneficial to H. cumingii and more feeding strategies of bioflocs in H. cumingii farming are expected to be developed in future.

Correction of substitution, deletion, and insertion mutations by 5′-tailed duplexes

Germline and somatic mutations cause various diseases, including cancer. Clinical applications of genome editing are keenly anticipated, since it can cure genetic diseases. Recently, we reported that a 5'-tailed duplex (TD), consisting of an approximately 80-base editor strand oligodeoxyribonucleotide and a 35-base assistant strand oligodeoxyribonucleotide, could edit a target gene on plasmid DNA and correct a single-base substitution mutation without an artificial nuclease in human cells. In this study, we assessed the ability of the TD to correct base substitution mutations located consecutively or separately, and deletion and insertion mutations. A TD with an 80-base editor strand was co-introduced into human U2OS cells with plasmid DNA bearing either a wild-type or mutated copepod green fluorescent protein (copGFP) gene. Among the mutations, three-base consecutive substitutions were efficiently repaired. The correction efficiencies of deletion mutations were similar to those of substitution mutations, and two to three times higher than those of insertion mutations. Up to three-base substitution, deletion, and insertion mutations were excellent targets for correction by TDs. These results suggested that the TDs are useful for editing disease-causing genes with small mutations.

Experimental genetic crosses in tsetse flies of the livestock pathogen Trypanosoma congolense savannah

BackgroundIn tropical Africa animal trypanosomiasis is a disease that has severe impacts on the health and productivity of livestock in tsetse fly-infested regions. Trypanosoma congolense savannah (TCS) is one of the main causative agents and is widely distributed across the sub-Saharan tsetse belt. Population genetics analysis has shown that TCS is genetically heterogeneous and there is evidence for genetic exchange, but to date Trypanosoma brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, with meiosis and production of haploid gametes. In T. brucei sex occurs in the fly salivary glands, so by analogy, sex in TCS should occur in the proboscis, where the corresponding portion of the developmental cycle takes place. Here we test this prediction using genetically modified red and green fluorescent clones of TCS.MethodsThree fly-transmissible strains of TCS were transfected with genes for red or green fluorescent protein, linked to a gene for resistance to the antibiotic hygromycin, and experimental crosses were set up by co-transmitting red and green fluorescent lines in different combinations via tsetse flies, Glossina pallidipes. To test whether sex occurred in vitro, co-cultures of attached epimastigotes of one red and one green fluorescent TCS strain were set up and sampled at intervals for 28 days.ResultsAll interclonal crosses of genetically modified trypanosomes produced hybrids containing both red and green fluorescent proteins, but yellow fluorescent hybrids were only present among trypanosomes from the fly proboscis, not from the midgut or proventriculus. It was not possible to identify the precise life cycle stage that undergoes mating, but it is probably attached epimastigotes in the food canal of the proboscis. Yellow hybrids were seen as early as 14 days post-infection. One intraclonal cross in tsetse and in vitro co-cultures of epimastigotes also produced yellow hybrids in small numbers. The hybrid nature of the yellow fluorescent trypanosomes observed was not confirmed by genetic analysis.ConclusionsDespite absence of genetic characterisation of hybrid trypanosomes, the fact that these were produced only in the proboscis and in several independent crosses suggests that they are products of mating rather than cell fusion. The three-way strain compatibility observed is similar to that demonstrated previously for T. brucei, indicating that a simple two mating type system does not apply for either trypanosome species.Graphical

Generation of a CAG-EGFP tagged cell line (KSCBi017-A-2) from human induced pluripotent stem cells using CRISPR/Cas9

Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.

Milk exosomes anchored with hydrophilic and zwitterionic motifs enhance mucus permeability for applications in oral gene delivery.

Exosomes have emerged as a promising tool for the delivery of drugs and genetic materials, owing to their biocompatibility and non-immunogenic nature. However, challenges persist in achieving successful oral delivery due to their susceptibility to degradation in the harsh gastrointestinal (GI) environment and impeded transport across the mucus-epithelium barrier. To overcome these challenges, we have developed high-purity bovine milk exosomes (mExo) as a scalable and efficient oral drug delivery system, which can be customized by incorporating hydrophilic and zwitterionic motifs on their surface. In our study, we observed significantly improved transport rates by 2.5-4.5-fold in native porcine intestinal mucus after the introduction of hydrophilic and zwitterionic surface modifications, as demonstrated by transwell setup and fluorescence recovery after photobleaching (FRAP) analysis. Remarkably, mExo functionalized by a block peptide (BP), consisting of cationic and anionic amino acids arranged in blocks at the two ends, demonstrated superior tolerability in the acidic gastric environment (with a protein recovery rate of 84.8 ± 7.7%) and exhibited a 2.5-fold increase in uptake by intestinal epithelial cells. Furthermore, both mExo and mExo-BP demonstrated successful intracellular delivery of functional siRNA, resulting in up to 65% suppression of the target green fluorescence protein (GFP) gene expression at a low dose of siRNA (5 pmol) without causing significant toxicity. These findings highlight the immense potential of modifying mExo with hydrophilic and zwitterionic motifs for effective oral delivery of siRNA therapies.

Inducible transgene expression in CHO cells using an artificial transcriptional activator with estrogen-binding domain.

Biopharmaceuticals, including therapeutic antibodies, are rapidly growing products in the pharmaceutical market. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are widely used as production hosts because recombinant antibodies require complex three-dimensional structures modified with sugar chains. Recombinant protein production using mammalian cells is generally performed with cell growth. In this study, we developed a technology that controls cell growth and recombinant protein production to induce recombinant protein production with predetermined timing. Expression of green fluorescent protein (GFP) gene and a single-chain antibody fused with the Fc-region of the human IgG1 (scFv-Fc) gene can be induced and mediated by the estrogen receptor-based artificial transcription factor Gal4-ERT2-VP16 and corresponding inducer drugs. We generated CHO cells using an artificial gene expression system. The addition of various concentrations of inducer drugs to the culture medium allowed control of proliferation and transgene expression of the engineered CHO cells. Use of 4-hydroxytamoxifen, an antagonist of estrogen, as an inducing agent yielded high gene expression at a concentration more than 10-fold lower than that of β-estradiol. When scFv-Fc was produced under inducing conditions, continuous production was possible for more than 2weeks while maintaining high specific productivity (57 pg cell-1 day-1 ). This artificial gene expression control system that utilizes the estrogen response of estrogen receptors can be an effective method for inducible production of biopharmaceuticals.

Long oligodeoxynucleotides: chemical synthesis, isolation via catching-by-polymerization, verification via sequencing, and gene expression demonstration.

Long oligodeoxynucleotides (ODNs) are segments of DNAs having over one hundred nucleotides (nt). They are typically assembled using enzymatic methods such as PCR and ligation from shorter 20 to 60 nt ODNs produced by automated de novo chemical synthesis. While these methods have made many projects in areas such as synthetic biology and protein engineering possible, they have various drawbacks. For example, they cannot produce genes and genomes with long repeats and have difficulty to produce sequences containing stable secondary structures. Here, we report a direct de novo chemical synthesis of 400 nt ODNs, and their isolation from the complex reaction mixture using the catching-by-polymerization (CBP) method. To determine the authenticity of the ODNs, 399 and 401 nt ODNs were synthesized and purified with CBP. The two were joined together using Gibson assembly to give the 800 nt green fluorescent protein (GFP) gene construct. The sequence of the construct was verified via Sanger sequencing. To demonstrate the potential use of the long ODN synthesis method, the GFP gene was expressed in E. coli. The long ODN synthesis and isolation method presented here provides a pathway to the production of genes and genomes containing long repeats or stable secondary structures that cannot be produced or are highly challenging to produce using existing technologies.

Somatic embryo production and GFP genetic transformation in elite Ugandan cassava genotypes

Friable embryogenic callus (FEC) production is a prerequisite for the genetic transformation of cassava (Manihot esculenta Crantz). This process necessitates the development of organized embryogenic structures (OES) as a precursor. However, the production of OES and FECs in cassava is dependent on the genotype. This study aimed to optimize somatic embryo production in elite Ugandan cassava genotypes (NASE13, NASE19, and NAROCASS1) and demonstrate genetic transformation using the enhanced green fluorescent protein (eGFP) gene. OES were induced by initiating leaf-lobe explants on Murashige and Skoog (MS) media at various concentrations of 24D and picloram followed by friable embryogenic callus (FEC) production and regeneration. There were highly significant differences among the different media compositions in the percentage response to OES formation (p < 0.001). NASE 13 exhibited the highest regeneration frequency of 38.4%; while NAROCASS1 had the lowest regeneration frequency of 10.5% recorded. The presence of GFP gene in NAROCASS1 was confirmed by polymerase chain reaction (PCR) analysis of the transformed lines. Compared to existing studies, this research pioneers genotype-specific optimization for somatic embryo production and successful genetic transformation in Ugandan cassava variaties. The utilization of these genotypes, already embraced by farmers, presents promising opportunities to enhance them with desirable traits, further augmenting cassava cultivation.