Development of Pichia pastoris as a rumen escape vehicle for the intestinal delivery of recombinant proteins in ruminants

The effectiveness of cellular encapsulation as a method for delivery of bioactive proteins and limiting amino acids to the small intestine of ruminants was investigated. Intracellular expression of green fluorescent protein variant (GFPuv) in Pichia pastoris was used as a visible marker to assess the cellular integrity of P. pastoris and determine the potential of this approach for protecting recombinant proteins from microbial proteolysis in the rumen. Fluorescent cells were easily identified in the presence of strained ruminal fluid when viewed by epifluorescent microscopy, and intact cells were readily enumerated. Batch cultures with rumen digesta demonstrated that 93, 97 and 25% of P. pastoris cells remained intact after 36 to 48 h of incubation in clarified ruminal fluid, an isolated bacterial fraction, and whole ruminal fluid, respectively. In continuous culture (Rusitec) with a dilution rate of 0.75 d-1, 19% of P. pastoris cells flowed intact from the fermentation vessels. In vitro abomasal simulations demonstrated that 84% of inoculated P. pastoris had lysed within 12 h of incubation, a property that is necessary for the release of encapsulated protein prior to the small intestine. These in vitro studies suggest that P. pastoris may be an effective vehicle for post-ruminal delivery of bioactive proteins in ruminants. Key words: Ruminal escape vehicle, Pichia pastoris, green fluorescent protein, bypass protein

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Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽

10.3390/v13040632 ◽

2021 ◽

Vol 13 (4) ◽

pp. 632

Author(s):

Yingyun Cai ◽

Shuiqing Yu ◽

Ying Fang ◽

Laura Bollinger ◽

Yanhua Li ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Lines ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Infectious Clone ◽

Hemorrhagic Fever ◽

Simian Hemorrhagic Fever Virus ◽

Hemorrhagic Fever Virus ◽

Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

Cell Transplantation ◽

10.1177/0963689720978219 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097821

Author(s):

Andrea Tenorio-Mina ◽

Daniel Cortés ◽

Joel Esquivel-Estudillo ◽

Adolfo López-Ornelas ◽

Alejandro Cabrera-Wrooman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Neural Induction ◽

Human Keratinocytes ◽

Differentiation Potential ◽

Neuronal Proteins ◽

Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

Applied and Environmental Microbiology ◽

10.1128/aem.01446-08 ◽

2008 ◽

Vol 74 (23) ◽

pp. 7431-7433 ◽

Cited By ~ 20

Author(s):

Mónica Martínez-Alonso ◽

Nuria González-Montalbán ◽

Elena García-Fruitós ◽

Antonio Villaverde

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Recombinant Proteins ◽

Fluorescent Protein ◽

Native Structure ◽

Functional Quality ◽

Soluble Aggregates ◽

Recombinant Polypeptides ◽

Green Fluorescent

ABSTRACT We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure.

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In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

Journal of Labelled Compounds and Radiopharmaceuticals ◽

10.1002/jlcr.25804401119 ◽

2001 ◽

Vol 44 (S1) ◽

pp. S339-S341

Author(s):

K. E. Luker ◽

G. D. Luker ◽

C. M. Pica ◽

J. L. Dahlheimer ◽

T. J. Fahrner ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Reporter Gene ◽

Fluorescent Protein ◽

Dual Function ◽

In Vivo Evaluation ◽

Green Fluorescent

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WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum

Physiological Genomics ◽

10.1152/physiolgenomics.00105.2005 ◽

2005 ◽

Vol 22 (3) ◽

pp. 412-421 ◽

Cited By ~ 19

Author(s):

Mira Wouters ◽

Karine Smans ◽

Jean-Marie Vanderwinden

Keyword(s):

Small Intestine ◽

Green Fluorescent Protein ◽

Gastrointestinal Tract ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Molecular Layer ◽

Transgenic Animals ◽

The Novel ◽

Suitable Alternative ◽

Green Fluorescent

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called WZsGreen. In the gastrointestinal tract of heterozygous WZsGreen/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel WZsGreen/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.

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Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽

10.1182/blood.v90.9.3304 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3304-3315 ◽

Cited By ~ 40

Author(s):

Marti F.A. Bierhuizen ◽

Yvonne Westerman ◽

Trudi P. Visser ◽

Wati Dimjati ◽

Albertus W. Wognum ◽

...

Keyword(s):

Bone Marrow ◽

Green Fluorescent Protein ◽

Gene Transfer ◽

Fluorescent Protein ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Egfp Expression ◽

Green Fluorescent ◽

Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

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The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2015.08.013 ◽

2015 ◽

Vol 256 ◽

pp. 22-29 ◽

Cited By ~ 6

Author(s):

Jo E. Lewis ◽

John M. Brameld ◽

Phil Hill ◽

Perry Barrett ◽

Francis J.P. Ebling ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Over Expression ◽

Green Fluorescent

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Recombinant Green Fluorescent Protein-Expressing Human Cytomegalovirus as a Tool for Screening Antiviral Agents

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.6.1588-1597.2000 ◽

2000 ◽

Vol 44 (6) ◽

pp. 1588-1597 ◽

Cited By ~ 101

Author(s):

Manfred Marschall ◽

Martina Freitag ◽

Sigrid Weiler ◽

Gabriele Sorg ◽

Thomas Stamminger

Keyword(s):

Green Fluorescent Protein ◽

Human Cytomegalovirus ◽

Fluorescent Protein ◽

Antiviral Agents ◽

Inhibitory Effects ◽

Plaque Reduction Assay ◽

Human Fibroblast Cultures ◽

Antiviral Activities ◽

Green Fluorescent

ABSTRACT A recombinant human cytomegalovirus (AD169-GFP) expressing green fluorescent protein was generated by homologous recombination. Infection of human fibroblast cultures with AD169-GFP virus produced stable and readily detectable amounts of GFP signals which were quantitated by automated fluorometry. Hereby, high levels of sensitivity and reproducibility could be achieved, compared to those with the conventional plaque reduction assay. Antiviral activities were determined for four reference compounds as well as a set of putative novel cytomegalovirus inhibitors. The results obtained were exactly in line with the known characteristics of reference compounds and furthermore revealed distinct antiviral activities of novel in vitro inhibitors. The fluorometric data could be confirmed by GFP-based flow cytometry and fluorescence microscopy. In addition, laboratory virus variants derived from the recombinant AD169-GFP virus provided further possibilities for study of the characteristics of drug resistance. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration- and time-dependent fashions and might also be adaptable for high-throughput screenings of cytomegalovirus-specific antiviral agents.

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432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab432 ◽

2010 ◽

Vol 22 (1) ◽

pp. 373

Author(s):

M. Reichenbach ◽

F. A. Habermann ◽

H. D. Reichenbach ◽

T. Guengoer ◽

F. Weber ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Germ Line ◽

Phosphoglycerate Kinase ◽

Healthy Male ◽

Transgenic Founder ◽

Green Fluorescent

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.

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