<ol><li> Der Abend vor der Transformation, Platten-293T-Zellen in 3-5x10 6 Zellen / 6 cm Gewebe cultureplate. Hinweis: Wir verwenden 293T-Zellen für ihre einfache Transfektion und efficacyas Virus-produzierenden Zellen. Diese Zellen weisen einen Mangel an Verpackung des Virus sei denn, ein Helfer-Plasmid eingeführt. </li><li> Am ersten Morgen, entfernen und ersetzen Sie mit 4 ml 293T Zelle med mit 25 mM Chloroquin. Legen Sie in Inkubator für 1 Stunde. Hinweis: Die Platte sollte ca. 80% konfluent. </li><li> In der Zwischenzeit bereiten Virus-machen Mischung für 2 Platten zu einer Zeit, in 5 ml Röhrchen: <ol><li> 2 x 10 mg retroviralen Plasmid-Konstrukt </li><li> 2 x 5 mg Verpackung Konstrukt (Ecopak) </li><li> 2 x 62 ml CaCl </li><li> komplette auf 1 ml mit sterilem ddH 2 O Hinweis: Während des retroviralen Plasmid kodiert das Onkogen von Interesse und ein Marker kodiert Ecopak gag-pol-env. Zusammen mit dem 293T-Zellen, werden sie erzeugen eine ecotropen Retrovirus (RV) spezifisch für Maus-Zellen, aber nicht infektiös für den menschlichen Zellen. Beide Chloroquin und CaCl 2 wurde gezeigt, dass die Transfektionseffizienz zu erhöhen. </li></ol></li><li> Add 1 ml 2x sterileHBS die Mischung tropfenweise, unter leichtem Vortexen der Röhre. </li><li> Unmittelbar fügen Sie diese Lösung, um 2 Platten, je 1 ml, sanft, Tropfen für Tropfen. </li><li> Legen Sie in Inkubator für 7 bis 11 Uhr. </li><li> Am Abend (7 bis 11 Uhr. Später), entfernen Medium, und sehr vorsichtig mit 5 ml frisch 293T Medium zu ersetzen. Legen Sie in Inkubator bis Mittag des nächsten Tages. Hinweis: Dieser Schritt ist wichtig, da man, um aus dem Chloroquin aus den Zellen benötigt. Wenn für längere Zeit gehalten werden, ist Chloroquin giftig. Die Lösung in den Platten sieht verschwommen, durch eine sehr feine, staubförmige Fällung der Transfektion Mischung. Hinweis: Es ist wichtig, dass alle Medien ändert sich mit extremen Milde zu tun, wie die Zellen wurden sensibilisiert durch die Chloroquin, und kann leicht von der Platte zu lösen. </li><li> Am nächsten Tag, 18 bis 24 Uhr. vor dem Abrufen des Virus (gegen Mittag), entfernen Medium, und sehr vorsichtig mit 3 ml frisch 293T Medium zu ersetzen. Ersetzen Sie in Inkubator O / N. </li><li> Der dritte Morgen (18 bis 24 Uhr. Höher), sanft saugen das Medium bilden die Platten mit einer 10 ml Spritze mit einer 18 G Nadel ausgestattet. Dieser Überstand enthält das Retrovirus. </li><li> Wechseln Sie die Nadel auf eine 45 mm Spritzenfilter, Invertzucker Spritze mehrmals, um die Lösung gut mischen, pass Überstand durch Filter und Aliquots in Kryoröhrchen Hinweis: Alternativ, wenn Sie machen 3 + Platten von Viren, Pool alle Überstände des gleichen Virus in eine 5 0 ml konischen Rohr. Gut mischen, dann aliquoten Überstände mit dem Retrovirus in Kryoröhrchen durch Filterung. </li><li> Das Röhrchen mit dem Virus-full Überstände werden in -80 ° C gehalten, bis verwendet. </li></ol>

<ol>
	<li>DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mutation+in+human+cells+by+using+an+Epstein-Barr+virus+shuttle+system.">Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.</a> Mol Cell Biol. 7, 379-387 (1987).</li><li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells.">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li><li>Finer, M. H., Dull, T. J., Qin, L., Farson, D., Roberts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=kat:+A+high-efficiency+retroviral+transduction+system+for+primary+human+T+lymphocytes.">kat: A high-efficiency retroviral transduction system for primary human T lymphocytes.</a> Blood. 83, 43-50 (1994).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+rapid+production+of+helper-free,+high-titer,+recombinant+retroviruses.">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> J Virol. 70, 5701-5705 (1996).</li><li>Pear, W. S., Miller, J. P., Xu, L., Pui, J. C., Soffer, B., Quackenbush, R. C., Pendergast, A. M., Bronson, R., Aster, J. C., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+induction+of+a+chronic+myelogenous+leukemia-like+myeloproliferative+disease+in+mice+receiving+P210+bcr/abl-transduced+bone+marrow.">Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.</a> Blood. 92, 3780-3792 (1998).</li><li>Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proc Natl Acad Sci USA. 90, 8392-8396 (1993).</li><li>Stocking, C., Kollek, R., Bergholz, U., Ostertag, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+terminal+repeat+sequences+impart+hematopoietic+transformation+properties+to+the+myeloproliferative+sarcoma+virus.">Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.</a> Proc Natl Acad Sci USA. 82, 5746-5750 (1985).</li><li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+chronic+myeloid+leukemia.">Models of chronic myeloid leukemia.</a> Curr Oncol Rep. 3, 228-237 (2001).</li><li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+pathogenesis+of+BCR-ABL++leukemia+in+mice.">Studying the pathogenesis of BCR-ABL+ leukemia in mice.</a> Oncogene. 21, 8643-8651 (2002).</li></ol>

Vier kritische Punkte sorgen für den Erfolg eines guten viralen Lager: <ol><li> Die 293T Zellen haben sich als sehr gesund, das heißt sie auf einer sehr regelmäßigen Abständen wurden aufgeteilt, wurden nie überwachsen und sind bei der optimierten Dichte von 3,5 bis 5.000.000 pro 6 cm Gewebekultur Platte ausplattiert, so dass sie eine Dichte erreichen von 80% der Platte am Morgen der Transfektion. </li><li> Die Zugabe von Chloroquin verbessert Transfektionseffizienz und anschließende Virustiter, ca. 3 - bis 5-fache, du...

Alle Reagenzien bei der Transfektion verwendet werden müssen sterilfiltriert (CaCl 2, 2x HBS, miliQ H 2 O) und steril gehalten.

production of replication-defective retrovirus by transient transfection of 293t cells

Unsere Laborstudien menschlichen myeloproliferativen Erkrankungen durch solche Onkogene als Bcr-Abl-oder Wachstumsfaktor-Rezeptor-derived Onkogene (ZNF198-FGFR1, Bcr-PDGFRα, etc.) induziert. Wir sind in der Lage zu modellieren und zu studieren eine dem Menschen ähnliche Erkrankung in unserem Mausmodell durch die Transplantation von Knochenmarkzellen zuvor mit einem Retrovirus Ausdruck des Onkogens von Interesse infiziert. Replikationsdefekte Retrovirus-Codierung ein menschliches Onkogen und ein Marker (GFP, RFP, Antibiotika-Resistenz-Gen, etc.) wird durch eine transiente Transfektion Protokoll mit 293T-Zellen, eine humane, renale epitheliale Zelllinie durch das Adenovirus E1A Genprodukt transformiert. 293-Zellen haben die ungewöhnliche Eigenschaft, sehr transfizierbare von Calciumphosphat (CaPO 4), mit bis zu 50-80% Transfektionseffizienz leicht erreichbar. Hier, Co-Transfektion wir 293-Zellen mit einem retroviralen Vektor, das Onkogen von Interesse und ein Plasmid, das die gag-pol-env Verpackung Funktionen, wie die Single-Genom Verpackung Konstrukte kat oder PCL, in diesem Fall die Ecopak Plasmid exprimiert. Die ersten Transfektion wird durch Verwendung von Chloroquin verbessert. Die Bestände an ecotropen Virus, wie Kulturüberstand 48 Stunden gesammelt. nach der Transfektion kann bei -80 ° C gelagert werden und zur Infektion von Zell-Linien im Hinblick auf die Transformation und in vitro-Untersuchungen oder primäre Zellen, wie zB Knochenmarkszellen der Maus, die dann für die Transplantation in unserem Mausmodell verwendet werden können .

Watch this Scientific Journal Video about Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells at JoVE.com

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Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Diese Technik zeigt eine effiziente Möglichkeit, replikationsdefekte retroviralen Aktien Kodierung eines menschlichen Onkogen vorzubereiten, und anschließend zur Induktion der myeloproliferativen Erkrankung im Mausmodell verwendet.

Produktion von replikationsdefekte Retrovirus durch transiente Transfektion von 293T-Zellen

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

production-replication-defective-retrovirus-transient-transfection

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JoVE Journal

Biology

19.4K Aufrufe.  Tufts University. Diese Technik zeigt eine effiziente Möglichkeit, replikationsdefekte retroviralen Aktien Kodierung eines menschlichen Onkogen vorzubereiten, und anschließend zur Induktion der myeloproliferativen Erkrankung im Mausmodell verwendet.

Serie: Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

Optimierte Transfektion Strategie für Expression und elektrophysiologische Ableitung von rekombinantem Voltage-Gated Ion Channels in HEK-293T-Zellen

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Forschung

Biologie

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Optimierung und Nutzung von<em&gt; Agrobacterium</em&gt; Transient-vermittelte Proteinproduktion in<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Transiente Expression von Proteinen durch Hydrodynamische Gentransfer in Mäusen

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Die Produktion von Haploid Zebrafischembryonen durch<em&gt; In-vitro-</em&gt; Düngung

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

High Yield Expression von rekombinanten humanen Proteinen mit der transiente Transfektion von HEK293 Zellen in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Vorübergehende Expression und zelluläre Lokalisation von rekombinanten Proteinen in gezüchteten Insektenzellen

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Untersuchung der Proteinexpression während der Entwicklung von Hühnerlinsen

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

Autor im Rampenlicht: Untersuchung der Linsenentwicklung und -funktion durch Mikroinjektion des RCAS(A)-Retrovirus in embryonaler Hühnerlinse 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

Mikroinjektion von rekombinantem RCAS(A)-Retrovirus in embryonale Hühnerlinse

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...