Generation Of Transgenic Lines Research Articles

Epigenetic Regulation of Endothelial Cell Lineages During Zebrafish Development-New Insights From Technical Advances.

Epigenetic regulation is integral in orchestrating the spatiotemporal regulation of gene expression which underlies tissue development. The emergence of new tools to assess genome-wide epigenetic modifications has enabled significant advances in the field of vascular biology in zebrafish. Zebrafish represents a powerful model to investigate the activity of cis-regulatory elements in vivo by combining technologies such as ATAC-seq, ChIP-seq and CUT&Tag with the generation of transgenic lines and live imaging to validate the activity of these regulatory elements. Recently, this approach led to the identification and characterization of key enhancers of important vascular genes, such as gata2a, notch1b and dll4. In this review we will discuss how the latest technologies in epigenetics are being used in the zebrafish to determine chromatin states and assess the function of the cis-regulatory sequences that shape the zebrafish vascular network.

Generation of Transgenic Lines of Zebrafish Expressing Fluorescently Tagged CCM Proteins to Study Their Function and Subcellular Localization Within the Vasculature.

Our knowledge of the structure, localization, and interaction partners of cerebral cavernous malformations (CCM) proteins is mainly based on cell culture studies that lack the physiology of a three-dimensional multi-tissue environment. Uncovering the subcellular localization and the dynamic behavior of CCM proteins is an important aspect of characterizing the endothelial cell biology of CCM scaffold formation and for describing interactions with other protein complexes. However, the generation of specific antibodies to locate CCM scaffolds within cells has been challenging. To overcome the lack of functional antibodies, here, we describe the methodology involved in the generation of a construct for the expression of a fluorescently labeled CCM fusion construct and in the establishment of a transgenic zebrafish reporter line. The transgenic expression of fluorescently labeled CCM proteins within the developing zebrafish vasculature makes it possible to study the detailed subcellular localization and the dynamics of CCM proteins in vivo.

The painted sea urchin, Lytechinus pictus, as a genetically-enabled developmental model.

Although sea urchins are one of the oldest and most widely used marine model systems, few species have been routinely kept in culture through multiple generations. The workhorse of the field is the purple urchin Strongylocentrotus purpuratus. However, one disadvantage of S. purpuratus is its long generation time, making it impractical as a model for generating and maintaining transgenic lines. In an effort to develop a sea urchin that is suitable for transgenerational experiments and the generation of transgenic lines, we have focused on development of updated culturing methods and genomic resources for the painted sea urchin, Lytechinus pictus. Compared to S. purpuratus, L. pictus have relatively large eggs, develop into optically clear embryos, and the smaller adults can become gravid in under a year. Fifty years ago, Hinegardner developed culturing methods for raising L. pictus through metamorphosis. Here, we provide an updated protocol for establishing and maintaining L. pictus in the laboratory, and describe a new genome resource for this urchin. In our hands, L. pictus reach the 4-armed pluteus stage at 4 days; become competent to metamorphosis at 24 days; and are gravid by 6 months. Plutei and juveniles are fed on a diet of algae and diatoms, and adults are fed on kelp. We also make available a L. pictus transcriptome generated from developmental stages (eggs to 2-day-old plutei) to support the annotation of our genome sequencing project, and to enhance the utility of this species for molecular studies and transgenesis.

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines.

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

Vectors for Genetically-Encoded Tags for Electron Microscopy Contrast in Drosophila

BackgroundOne of the most notable recent advances in electron microscopy (EM) was the development of genetically-encoded EM tags, including the fluorescent flavoprotein Mini-SOG (Mini-Singlet Oxygen Generator). Mini-SOG generates good EM contrast, thus providing a viable alternative to technically-demanding methods such as immuno-electron microcopy (immuno-EM). Based on the Mini-SOG technology, in this paper, we describe the construction, validation and optimization of a series of vectors which allow expression of Mini-SOG in the Drosophila melanogaster genetic model system.FindingsWe constructed a Mini-SOG tag that has been codon-optimized for expression in Drosophila (DMS tag) using PCR-mediated gene assembly. The photo-oxidation reaction triggered by DMS was then tested using these vectors in Drosophila cell lines. DMS tag did not affect the subcellular localization of the proteins we tested. More importantly, we demonstrated the utility of the DMS tag for EM in Drosophila by showing that it can produce robust photo-oxidation reactions in the presence of blue light and the substrate DAB; the resultant electron micrographs contain electron-dense regions corresponding to the protein of interest. The vectors we generated allow protein tagging at both termini, for constitutive and inducible protein expression, as well as the generation of transgenic lines by P-element transformation.ConclusionsWe demonstrated the feasibility of Mini-SOG tagging in Drosophila. The constructed vectors will no doubt be a useful molecular tool for genetic tagging to facilitate high-resolution localization of proteins in Drosophila by electron microscopy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0034-1) contains supplementary material, which is available to authorized users.

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

SummaryTools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has been shown to hold significant promise as a production platform for recombinant proteins, but transformation of the nuclear genome is still a non-trivial process due to random gene insertion and frequent silencing. Insertion of transgenes into the chloroplasts is an alternative strategy, and we report here the stable expression of a large (91 kDa) protein in the chloroplast using a recently developed low-cost transformation protocol. Moreover, selection of transformants is based on restoration of prototrophy using an endogenous gene (psbH) as the marker, thereby allowing the generation of transgenic lines without the use of antibiotic-resistance genes. Here, we have expressed a bifunctional diterpene synthase in C. reinhardtii chloroplasts. Homoplasmic transformants were obtained with the expressed enzyme accounting for 3.7 % of total soluble protein. The enzyme was purified to homogeneity and expression was shown to have a small but reproducible effect on growth rate at the end of log phase growth. These results demonstrate that large recombinant enzymes can be synthesised in the algal chloroplast, and serve to underline its potential as a platform for the biosynthesis of novel metabolites.

Transgenic animal models for the production of human immunocompounds in milk to prevent diarrhea, malnourishment and child mortality: perspectives for the Brazilian Semi-Arid region

Diarrhea is one of the most significant issues for global health in children under the age of five. Close to 60% of mortality in this risk group is caused by pneumonia, diarrhea or measles, which is usually associated with malnutrition. In Brazil, this scenario is no different, with the Northeast semi-arid region having alarming rates of child mortality, almost twice that of the current national mortality rate, with some cities being three times higher, placing child mortality rates in the Northeast of Brazil among those of the most problematic in the world. The oral rehydration solution, introduced by the World Health Organization (WHO), is seen as one of the major medical advances in the past 50 years, and is believed to save the lives of 1 to 2 million children each year. However, the oral rehydration solution simply restores body fluid normality, treating the consequences and not the causes of diarrheas. Other means to effectively address worldwide diarrheic ailments still need to be devised or improved, focusing more on preventive rather than curative approaches, to prevent or shorten the course of disease and to minimize recurrences. A great example is breast-feeding. A series of studies documented the reduction of diarrhea episodes in breast-fed children and, also, noted a faster recovery time following an infection in these children. The positive benefit of breast milk is attributed to the antimicrobial actions of human milk proteins, such as lysozyme and lactoferrin that can enhance intestinal and systemic immunological functions. Unfortunately, breast-feeding is not always an option, especially for toddlers, which is usually aggravated by undernourishment and low standard of living, reinforcing the need for alternative strategies. Consequently, to successfully combat childhood undernourishment, morbidity and mortality in the Northeast region of Brazil, the investment in approaches and models that are more suitable to the local semi-arid adapted agriculture-base is required. The goat is an important economic asset to the Northeast region, providing milk, meat and leather, with the number of goats in the Northeast region representing more than 90% of the Brazilian herd. Historically, goats have been used as a model in biotechnological studies involving the expression of specific proteins in the milk of lactating animals. In this way, the use of goats expressing recombinant human lysozyme (rhLZ) and lactoferrin (rhLF) in milk is a proven reality that can be employed for the reduction of malnutrition, infectious diseases and diarrhea in children in the Brazilian semi-arid region with an extraordinary potential for success. Due to the protective nature of human milk imparted, in part, by the presence of lysozyme and lactoferrin, the generation of transgenic lines for the production of these important antimicrobial compounds in the milk of the local goat population is a unique approach for delivering the potential protective benefits of human milk. In addition, the use of rhLZ and rhLF transgenic animals can be used as a research tool for studying and elucidating the individual roles of these antimicrobial proteins in gut development and their synergistic in vivo effects. The proposed research was designed with the rational that the transgenic approach can be used to modify the milk composition of dairy animals to supply milk-borne human immunocompounds (rhLZ, rhLF) and nutrients University of Fortaleza, UNIFOR, Fortaleza, Brazil Full list of author information is available at the end of the article Bertolini et al. BMC Proceedings 2014, 8(Suppl 4):O30 http://www.biomedcentral.com/1753-6561/8/S4/O30

The ETRAMP Family Member SEP2 Is Expressed throughout Plasmodium berghei Life Cycle and Is Released during Sporozoite Gliding Motility

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.

UJI KETAHANAN GALUR-GALUR KENTANG TRANSGENIK HASIL TRANSFORMASI DENGAN GEN RB TERHADAP PENYAKIT HAWAR DAUN (PHYTOPHTHORA INFESTANS) DI KP PASIRSARONGGE, CIANJUR

Resistance test strains of transgenic potatoes transformed with RB gene to late blight (Phytophthora infestan) in KP Pasirsarongge, Cianjur. Potato late blight caused by Phytophthora infestans (P. infestans) (Mönt.) de Barry continues to be one of the most important crop diseases of all time. Genetic engineering of potato using RB gene for resistant plant to this disease is the most effective and environmental friendly to prevent widespread of late blight. This research aims to perform resistance of transgenic potato lines containing RB gene to lateblight (P. infestans) in Pasirsarongge, Cianjur field trial station. The first generation of transgenic lines were planted on polybag containing soil:manures using randomized complete block design. Tested plant inoculation was done naturaly from inoculum source from border row (Granola) that has been planted at one month before. The symptom was observed at one month after planting and damage scoring was done every three days for five times. Twenty two transgenic lines of tested plant showed various resistance respond to late blight (P. infestans) attack. Three transgenic lines showed highly resistance to late blight (P. infestans) were lines 11, 24, and 25, one transgenic line has resistant level was line 6.

An in vivo Transient Expression System Can Be Applied for Rapid and Effective Selection of Artificial MicroRNA Constructs for Plant Stable Genetic Transformation

The utility of artificial microRNAs (amiRNAs) to induce loss of gene function has been reported for many plant species, but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable. In this study, expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation. A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments. Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs. This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.

Transposon‐mediated transgenesis, transgenic rescue, and tissue‐specific gene expression in rodents and rabbits

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

Characterization of a novel type I keratin gene and generation of transgenic lines with fluorescent reporter genes driven by its promoter/enhancer in Xenopus laevis

We investigated the characteristics of a novel type I keratin gene in Xenopus laevis during ontogenesis. The transcript was first detected in the posterior region at the late neurula stage, and then restricted to the fin and external gill during embryogenesis. To examine the transcriptional regulation of the keratin gene in vivo, we generated transgenic lines with fluorescent reporter genes driven by its 4.2-kb upstream sequence. The promoter/enhancer activity recapitulated the endogenous gene expression during embryogenesis. Sequential deletion analyses revealed that the regions proximal to the promoter were essential for fin-specific expression. Reporter expression was detected in various organs, including the fin and gill. In particular, robust expression was observed in the developing limbs and gill. The reporter fluorescence rapidly decreased with internal gill resorption during metamorphosis. The transgenic lines carrying the promoter/enhancer should represent valuable tools for elucidating the formation, development and resorption of various organs, especially the gill.

Neutrophil-selective CD18 silencing using RNA interference in vivo

AbstractTissue-specific silencing of genes may be used for genetic engineering in mice and has possible therapeutic applications in humans. Current strategies in mice rely on Cre/loxP technology requiring the generation of multiple transgenic lines and breeding strategies. Here, we describe the selective silencing of CD18, a leukocyte-specific integrin in neutrophils using a micro RNA (miRNA) strategy that requires the generation of one transgenic line. CD18-specific miRNA hairpin driven by the myeloid specific human MRP8 promoter resulted in the generation of transgenic lines with 75% to 95% reduction in CD18 protein levels in neutrophils and monocytes. Minimal decreases in T cells and a partial diminution in macrophages were observed. Neutrophil CD18 silencing resulted in neutrophilia, splenomegaly, and significant defects in neutrophil trafficking with the degree of alterations correlating with the extent of CD18 silencing. Thus, our data demonstrate the utility of using miRNA approaches to silence genes in neutrophils, which are terminally differentiated cells with a short half-life that largely precludes their genetic manipulation in vitro. Furthermore, the mouse models provide a valuable tool to examine the contribution of CD18 on neutrophils to leukocyte adhesion deficiency type I (LAD-I), a complex inherited disorder in which reduced or absent CD18 expression in multiple leukocyte subsets leads to impaired innate and adaptive immune responses.

Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR

The generation of transgenic lines is vital to many genetic strategies and provides useful reagents for cell labeling and lineage-tracing experiments. Transposon-based systems offer simple, yet robust, platforms for transgenesis in the frog. Here, we provide a protocol for a microinjection-based transposon transgenesis method using a 'natural breeding' strategy for the collection of Xenopus tropicalis embryos. This method uses co-injection of a plasmid containing a transposon substrate together with synthetic mRNA encoding the transposase to achieve efficient integration of the transgene in the frog genome. We also describe a modified extension primer tag selection linker-mediated PCR technique to identify transposon integration sites within the host genome. This cloning strategy allows rapid identification of genomic sequences flanking the integration sites and multiple independently segregating transposon integration events in a single tadpole can be cloned simultaneously.

REDUCTION OF TOTAL STEROIDAL GLYCOALKALOIDS IN POTATO TUBERS USING ANTISENSE CONSTRUCTS OF A GENE ENCODING A SOLANIDINE GLUCOSYL TRANSFERASE

REDUCTION OF TOTAL STEROIDAL GLYCOALKALOIDS IN POTATO TUBERS USING ANTISENSE CONSTRUCTS OF A GENE ENCODING A SOLANIDINE GLUCOSYL TRANSFERASE

Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system.

The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.