scholarly journals Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa

2018 ◽  
Author(s):  
Kerry Swartwood ◽  
Joyce Van Eck
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Genetic Engineering ◽  
Large Scale ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Scale Production ◽  
Hypocotyl Explants

AbstractPhysalis pruinosa, also known as groundcherry, produces a small, yellow, highly nutritious edible fruit that is enveloped by a papery husk. In order for the potential of large-scale production of P. pruinosa fruit to be realized, undesirable characteristics, such as an unmanageable, sprawling growth habit and extensive fruit drop, need to be improved by exploiting approaches available through plant breeding, genetic engineering, and gene editing. In this study, we established plant regeneration and Agrobacterium tumefaciens-mediated methods to allow application of genetic engineering and gene editing of P. pruinosa. Cotyledon and hypocotyl explants from 7 – 8-day-old in vitro-grown seedlings were assessed for plant regeneration. Explants were cultured for 2 weeks on a Murashige and Skoog salts-based medium that contained 2 mg/L zeatin followed by transfer to medium containing 1 mg/L zeatin. Only hypocotyl explants regenerated shoots. Hypocotyl explants were infected with Agrobacterium tumefaciens strain AGL1 containing the pJL33 binary vector that has the green fluorescent protein (GFP) reporter and neomycin phosphotransferase II (nptII) selectable marker genes. After cocultivation, explants were cultured on selective plant regeneration medium that contained 50, 100, 200, 250, and 300 mg/L kanamycin to determine the most effective level for efficient recovery of transgenic lines. Based on rooting of regenerated shoots on selective medium, GFP visualization, and PCR analysis for the presence of the nptII gene, medium containing 200 mg/L kanamycin resulted in the highest transformation efficiency at 24%. This study sets the foundation for future genetic engineering and gene editing approaches for improvement of P. pruinosa.

scholarly journals Cannabis sativa: From Therapeutic Uses to Micropropagation and Beyond

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2078
Author(s):  
Tristan K. Adams ◽  
Nqobile A. Masondo ◽  
Pholoso Malatsi ◽  
Nokwanda P. Makunga
Keyword(s):  
Tissue Culture ◽  
Genetic Engineering ◽  
Large Scale ◽  
Gene Editing ◽  
Cannabis Sativa ◽  
Scale Production ◽  
Indirect Organogenesis ◽  
Large Scale Production ◽  
Pharmaceutical Industries

The development of a protocol for the large-scale production of Cannabis and its variants with little to no somaclonal variation or disease for pharmaceutical and for other industrial use has been an emerging area of research. A limited number of protocols have been developed around the world, obtained through a detailed literature search using web-based database searches, e.g., Scopus, Web of Science (WoS) and Google Scholar. This article reviews the advances made in relation to Cannabis tissue culture and micropropagation, such as explant choice and decontamination of explants, direct and indirect organogenesis, rooting, acclimatisation and a few aspects of genetic engineering. Since Cannabis micropropagation systems are fairly new fields, combinations of plant growth regulator experiments are needed to gain insight into the development of direct and indirect organogenesis protocols that are able to undergo the acclimation stage and maintain healthy plants desirable to the Cannabis industry. A post-culture analysis of Cannabis phytochemistry after the acclimatisation stage is lacking in a majority of the reviewed studies, and for in vitro propagation protocols to be accepted by the pharmaceutical industries, phytochemical and possibly pharmacological research need to be undertaken in order to ascertain the integrity of the generated plant material. It is rather difficult to obtain industrially acceptable micropropagation regimes as recalcitrance to the regeneration of in vitro cultured plants remains a major concern and this impedes progress in the application of genetic modification technologies and gene editing tools to be used routinely for the improvement of Cannabis genotypes that are used in various industries globally. In the future, with more reliable plant tissue culture-based propagation that generates true-to-type plants that have known genetic and metabolomic integrity, the use of genetic engineering systems including “omics” technologies such as next-generation sequencing and fast-evolving gene editing tools could be implemented to speed up the identification of novel genes and mechanisms involved in the biosynthesis of Cannabis phytochemicals for large-scale production.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Xuan Guan ◽  
David L Mack ◽  
Claudia M Moreno ◽  
Fernando Santana ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Urine ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Cellular Reprogramming ◽  
Urine Samples ◽  
Pcr Analysis ◽  
Scale Production ◽  
Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

scholarly journals Yeast Actin-Related Protein ARP6 Negatively RegulatesAgrobacterium-Mediated Transformation of Yeast Cell

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Yumei Luo ◽  
Zikai Chen ◽  
Detu Zhu ◽  
Haitao Tu ◽  
Shen Quan Pan
Keyword(s):  
Genetic Engineering ◽  
Large Scale ◽  
Transformation Efficiency ◽  
Genetic Diseases ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Scale Production ◽  
Related Protein ◽  
Cellular Mechanisms ◽  
Eukaryotic Organisms

The yeasts, includingSaccharomyces cerevisiaeandPichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values.Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related proteinARP6as a negative regulator of AMT.ARP6is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting thatARP6might regulate AMT via SWR-C. Moreover, knockout ofARP6led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

scholarly journals Application of RNA Interference Technology to Acroporid Juvenile Corals

2021 ◽  
Vol 8 ◽  
Author(s):  
Ikuko Yuyama ◽  
Tomihiko Higuchi ◽  
Michio Hidaka
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Stress Response ◽  
Large Scale ◽  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Green Fluorescent

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

204 Efficient editing of porcine parthenogenetic zygotes by electroporation of Cas9 ribonucleoprotein complexes

2019 ◽  
Vol 31 (1) ◽  
pp. 227
Author(s):  
F. L. Ongaratto ◽  
P. Rodriguez-Villamil ◽  
U. Ganbaatar ◽  
C. De Frutos ◽  
S. Solin ◽  
...  
Keyword(s):  
Large Scale ◽  
Gene Editing ◽  
The Other ◽  
Scale Production ◽  
Efficient System ◽  
Ribonucleoprotein Complexes ◽  
Group 4 ◽  
Group 2 ◽  
And Control ◽  
Group 1

Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250ng μL−1 of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n=130): 20V, 3ms, ×2 pulses, 1 repeat; group 2 (n=102): 20V, 1ms, ×2 pulses, 2 repeats; and Control (n=96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250ng μL−1 of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n=17): 20V, 3ms, ×1 pulses, 1 repeat; group 2 (n=49): 20V, 3ms, ×3 pulses, 1 repeat; group 3 (n=64): 30V, 3ms, ×1 pulses, 1 repeat; group 4 (n=61): 30V, 3ms, ×3 pulses, 1 repeat; group 5 (n=120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25ng μL−1), and Control (n=76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P<0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P<0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P<0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P<0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.

scholarly journals Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5104-5110 ◽  
Author(s):  
Robert E. Throm ◽  
Annastasia A. Ouma ◽  
Sheng Zhou ◽  
Anantharaman Chandrasekaran ◽  
Timothy Lockey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Cell Clone ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Scale Production ◽  
Producer Cell

AbstractRetroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

scholarly journals A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Xudong Wu ◽  
Di Wu ◽  
Zhisheng Lu ◽  
Wentao Chen ◽  
Xiaojian Hu ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scale Production ◽  
Sequence Specificity ◽  
Tev Protease ◽  
Large Scale Production ◽  
Novel Method

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants ofsfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant ofsfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. ThesfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.

scholarly journals Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Donald J. Tipper ◽  
Eva Szomolanyi-Tsuda
Keyword(s):  
Yeast Cell ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Subcutaneous Administration ◽  
Polyoma Virus ◽  
Apolipoprotein A1 ◽  
Yeast Cells ◽  
Scale Production ◽  
Protein Antigens ◽  
Virus Challenge

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposesβ-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use.Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

scholarly journals Gene Profiles and Electrophysiology of Doublecortin-Expressing Cells in the Subventricular Zone after Ischemic Stroke

2008 ◽  
Vol 29 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Xian Shuang Liu ◽  
Michael Chopp ◽  
Xue Guo Zhang ◽  
Rui Lan Zhang ◽  
Ben Buller ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Subventricular Zone ◽  
Fluorescent Protein ◽  
Marker Gene ◽  
Brain Slices ◽  
Resting Membrane Potential ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Neuronal Marker ◽  
Gene Profiles

Stroke increases neuroblasts in the subventricular zone (SVZ) of the lateral ventricle and these neuroblasts migrate toward the ischemic boundary to replace damaged neurons. Using brain slices from the nonischemic adult rat and transgenic mice that expressed enhanced green fluorescent protein (EGFP) concomitantly with doublecortin (DCX), a marker for migrating neuroblasts, we recorded electrophysiological characteristics while simultaneously analyzing the gene expression in single SVZ cells. We found that SVZ cells expressing the DCX gene from the nonischemic rat had a mean resting membrane potential (RMP) of −30 mV. DCX—EGFP-positive cells in the nonischemic SVZ of the transgenic mouse had a mean RMP of −25 ± 7 mV and did not exhibit Na+ currents, characteristic of immature neurons. However, DCX—EGFP-positive cells in the ischemic SVZ exhibited a hyperpolarized mean RMP of −54 ± 18 mV and displayed Na+ currents, indicative of more mature neurons. Single-cell multiplex RT-PCR analysis revealed that DCX—EGFP-positive cells in the nonischemic SVZ of the transgenic mouse expressed high neural progenitor marker genes, Sox2 and nestin, but not mature neuronal marker genes. In contrast, DCX—EGFP-positive cells in the ischemic SVZ expressed tyrosine hydroxylase, a mature neuronal marker gene. Together, these data indicate that stroke changes gene profiles and the electrophysiology of migrating neuroblasts.

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