Diese Arbeit wurde von den National Institutes of Health (7R01HL098528-03) und FAMRI Clinical Innovator Award unterstützt.

1. Herstellung von Protein-Transfektionsreagenz <ol><li> Lösen Sie die Pro-Ject Reagenz durch Zugabe von 250 ul Methanol oder Chloroform zu dem Röhrchen mit dem trockenen Film. </li><li> Vortex für 10-20 sec bei Höchstgeschwindigkeit. </li><li> Je 20 ul Pro-Ject Reagenz in separaten Reaktionsgefäßen. </li><li> Verdampft das Lösungsmittel, indem die Reaktionsgefäße mit dem Pro-Ject Reagenz unter einer Laminar-Flow-Haube für mindestens 6 h bei Raumtemperatur. Es muss vollständig trocken sein. Alternativ trocknen die Pro-Ject Reagens unter Verwendung einer Vakuum getrocknet. </li><li> Bewahren Sie die Röhrchen bei -20 ° C. Sie sind gut für ein Jahr bei dieser Temperatur. </li></ol> 2. Hinzufügen Protein zu Pro-Ject Reagenz <ol><li> Je 50 ul PBS, enthaltend 2,0 ug des Proteins von Interesse, in ein Röhrchen mit 20 ul getrockneten Pro-Ject Transfektionsreagenz. </li><li> Nach Zugabe des Proteins, mischen Sie die Lösung durch Vortexen bei niedriger Geschwindigkeit foder 3-5 sec, gefolgt von Inkubation für 30 min bei Raumtemperatur. </li></ol> 3. Intratracheale Verwaltung von Protein <ol><li> Anesthetize Mäuse durch intraperitoneale Injektion von Ketamin / Xylazin (100 mg / kg und 10 mg / kg). </li><li> Nach Sedierung erreicht ist, werden die Mäuse aus ihren oberen Schneidezähne mit einem speziell entwickelten Plattform (Abbildung 1) suspendiert. </li><li> Mit einer Pinzette und einer Büroklammer nach einem Laryngoskop wird die Oropharynx geöffnet, so daß der Katheter eingeführt werden kann (Abb. 2). Die Stimmbänder sind visualisiert, indem eine Lichtquelle gegen die Luftröhre der Maus. </li><li> Das Protein / Projekt Reagenzmischung (50 ul) wird durch die Luftröhre mit einer Penn Century Microsprayer injiziert. </li></ol>

<ol>
	<li>Glasser, S. W., Korfhagen, T. R., Wert, S. E., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+models+for+study+of+pulmonary+development+and+disease.">Transgenic models for study of pulmonary development and disease.</a> Am. J. Physiol. 267, 489-497 (1994).</li><li>D'Armiento, J., Dalal, S. S., Okada, Y., Berg, R. A., Chada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+expression+in+the+lungs+of+transgenic+mice+causes+pulmonary+emphysema.">Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema.</a> Cell. 71, 955-961 (1992).</li><li>Foronjy, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+expression+attenuates+cigarette+smoke-+or+elastase-generated+emphysema+in+mice.">Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice.</a> Am. J. Respir Crit. Care Med. 173, 623-631 (2006).</li><li>Foronjy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+divergent+roles+of+secreted+frizzled+related+protein-1+(SFRP1)+in+lung+morphogenesis+and+emphysema.">The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema.</a> Am. J. Pathol. 177, 598-607 (2010).</li><li>Costa, R. H., Kalinichenko, V. V., Lim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+in+mouse+lung+development+and+function.">Transcription factors in mouse lung development and function.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 280, 823-838 (2001).</li><li>Perl, A. K., Tichelaar, J. W., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+the+respiratory+epithelium+of+the+mouse.">Conditional gene expression in the respiratory epithelium of the mouse.</a> Transgenic Research. 11, 21-29 (2002).</li><li>Morimoto, M., Kopan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rtTA+toxicity+limits+the+usefulness+of+the+SP-C-rtTA+transgenic+mouse.">rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse.</a> Dev. Biol. 325, 171-178 (2009).</li><li>Waehler, R., Russell, S. J., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+targeted+viral+vectors+for+gene+therapy.">Engineering targeted viral vectors for gene therapy.</a> Nature reviews. Genetics. 8, 573-587 (2007).</li><li>Crystal, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+an+adenovirus+containing+the+human+CFTR+cDNA+to+the+respiratory+tract+of+individuals+with+cystic+fibrosis.">Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.</a> Nat. Genet. 8, 42-51 (1994).</li><li>Merkel, O. M., Zheng, M., Debus, H., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+gene+delivery+using+polymeric+nonviral+vectors.">Pulmonary gene delivery using polymeric nonviral vectors.</a> Bioconjugate Chemistry. 23, 3-20 (2012).</li><li>Yang, Y., Li, Q., Ertl, H. C., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+humoral+immune+responses+to+viral+antigens+create+barriers+to+lung-directed+gene+therapy+with+recombinant+adenoviruses.">Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.</a> J. Virol. 69, 2004-2015 (1995).</li><li>Liu, Q., Muruve, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+inflammatory+response+to+adenovirus+vectors.">Molecular basis of the inflammatory response to adenovirus vectors.</a> Gene Ther. 10, 935-940 (2003).</li><li>Pfeifer, C., Aneja, M. K., Hasenpusch, G., Rudolph, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotype+9-mediated+pulmonary+transgene+expression:+effect+of+mouse+strain,+animal+gender+and+lung+inflammation.">Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation.</a> Gene Ther. 18, 1034-1042 (2011).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+regulates+innate+immune+and+proteolytic+responses+to+cigarette+smoke+exposure+in+the+lung.">Protein phosphatase 2A regulates innate immune and proteolytic responses to cigarette smoke exposure in the lung.</a> Toxicol. Sci. 126, 589-599 (2012).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphatase+2a+(Pp2a)+Regulates+Innate+Immune+and+Proteolytic+Responses+to+Cigarette+Smoke+Exposure+in+the+Lung.">Protein Phosphatase 2a (Pp2a) Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung.</a> Toxicol. Sci. , (2012).</li><li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J. Appl. Physiol. 87, 2362-2365 (1999).</li></ol>

Die Autoren haben keine Interessenkonflikte offen zu legen.

Der Vorteil dieser Technik gegenüber anderen Methoden ist, dass sie in Protein-Spiegel erhöht und die Aktivität im Lungengewebe selbst erzeugt. Darüber hinaus dringt zu den distalen Bereichen der Lunge zu bleiben nur innerhalb der Atemwege gegenüber. Wir haben erhöhte Protein-Aktivität unserer Injektat auch nach lavaging der Atemwege mit einer Salzlösung 15 gemessen. Tissue Aktivität Analysen zeigen, dass das Protein in Zellen gelangen und nicht nur in den Lufträumen der Maus bleiben. Dies wurde durch Immunhistochemie zeigt diffuse Färbung der injizierten Proteins in den Alveolen und Atemwege der Maus (3) bestätigt. Wichtig ist, dass dieses Protokoll nicht provozieren Lungenentzündung oder Protease Ausdruck. So können alle Änderungen, die auftreten, zu tun, um die Auswirkungen der verabreichten Protein zugeschrieben werden. Das Fehlen von entzündlichen Reaktion ermöglicht die Verwendung von wiederholten Injektionen Wirkungen über einen Longe prüfenr Zeitraum.

<table><tbody><tr><td>Pro-Ject</td><td>Pierce Bio</td><td>89850</td><td></td></tr><tr><td>Microsprayer</td><td>Penn Century</td><td>FMJ-250</td><td></td></tr><tr><td>Beta-galactosidase</td><td>Pierce Bio</td><td>89850</td><td></td></tr><tr><td>Beta-galactosidase antibody</td><td>Santa Cruz Bio</td><td>SC-19119</td><td></td></tr><tr><td>Mouse serum albumin</td><td>Sigma Aldrich</td><td>A3139</td><td></td></tr><tr><td>Ketamine/xylazine</td><td>Sigma Aldrich</td><td>K113</td><td></td></tr></tbody></table>

protein transfection of mouse lung

Zunehmende Proteinexpression ermöglicht es Forschern, besser zu verstehen, die funktionelle Rolle dieses Proteins bei der Regulation biologischer Prozesse Taste 1. In der Lunge wurde dies typischerweise durch genetische Ansätze transgenen Mäusen 2,3 oder virale oder nicht-virale Vektoren, die Protein-Ebene über eine erhöhte Genexpression 4 erhöhen nutzen erreicht. Transgene Mäuse sind teuer und zeitaufwendig zu erzeugen und die zufällige Insertion eines Transgens oder chronischen Genexpression normale Entwicklung der Lunge zu ändern und so die Nutzbarkeit des Modells 5. Während bedingten transgenics abzuwenden Probleme mit chronischer Genexpression 6 der reversen Tetracyclin-gesteuerten Transaktivator (rtTA)-Mäuse, die zur bedingten Ausdruck erzeugen zugeordnet sind, zu entwickeln spontanes Zwischenraumvergrößerung 7. Wie bei transgenen, ist die Verwendung von viralen und nicht-viralen Vektoren teuer 8 und Dosis-d provozierenependent Entzündungsreaktionen dass verwirren Ergebnisse 9 und 10 behindern Ausdruck. Darüber hinaus sind die Wirksamkeit von wiederholten Dosen durch verstärkte Immunantwort auf den Vektor 11,12 begrenzt. Forscher entwickeln Adeno-assoziierter viraler (AAV) Vektoren, die weniger Entzündung zu provozieren und mehr Ausdruck in der Lunge 13. Mit β-Galactosidase, präsentieren wir eine Methode zur schnellen und effektiv erhöht Proteinexpression in der Lunge über eine direkte Protein Transfektionstechnik. Dieses Protokoll mischt eine feste Menge an gereinigtem Protein mit 20 ul einer Lipid-basierte Transfektionsreagenz (Pro-Ject, Pierce Bio), um das Eindringen in das Lungengewebe selbst zu ermöglichen. Die liposomale Proteinmischung wird anschließend in den Lungen der Mäuse über die Luftröhre mit einem Microsprayer (Penn Century, Philadelphia, PA) injiziert. Die Feinzerstäuber erzeugt eine feine Wolke aus flüssigen Aerosol in den Lungen. Mit Hilfe der Technikwir haben eine gleichmäßige Abscheidung des injizierten Proteins während der Atemwege und die Alveolen von Mäusen 14 demonstriert. Das Lipid Transfektion Technik ermöglicht die Verwendung einer kleinen Menge an Protein zu erzielen. Dies begrenzt die entzündliche Reaktion, die ansonsten durch hohe Protein Verabreichung hervorgerufen würde. In der Tat, mit dieser Technik, die wir veröffentlicht, dass wir in der Lage, deutlich zu erhöhen PP2A Aktivität in der Lunge, ohne die Lungenspülung Zellularität 15 waren. Lungenspülung Zellularität genommen 24 Stunden nach der Belastung war vergleichbar mit Kontrollen (27 ± 4 Kontrolle vs 31 ± 5 Albumin transfizierten; N = 6 pro Gruppe). Außerdem erhöht es Protein-Ebene ohne eine Lunge entwicklungsbedingten Veränderungen oder Änderungen an der Architektur, die in transgenen Modellen auftreten können. Jedoch kann die Notwendigkeit einer wiederholten Verabreichungen machen diese Technik weniger günstig für Studien, die die Auswirkungen einer langfristigen Erhöhung der Proteinexpression. Dies würde besonders tru seine für Proteine ​​mit kurzen Halbwertszeiten.

<html> Um die Wirksamkeit unserer Technik demonstrieren verwendeten wir eine Feinzerstäuber (Penn Jahrhundert), um die Luftröhre von Mäusen mit 2 ug Maus Albumin (Sigma) in 50 ul PBS, das 20 ul Pro-Ject Transfektionsreagenz enthielt, gelöst zu injizieren. Die Albumin behandelten Mäusen mit Mäusen, die in gleicher Weise mit 2 ug beta-Galaktosidase-Proteins (Pierce Bio) behandelt wurden, verglichen. Nach 24 Stunden wurden die Mäuse getötet und die Lungen wurden für die histologische Analyse verarbeitet. Immunhistochemie für beta-Galactosidase wurde mit Formalin fixierten Lungengewebe Abschnitte von dem Albumin und beta-Galactosidase-behandelten Mäusen durchgeführt. Selbst 24 Stunden nach der Transfektion von Proteinen, wiesen wir eine intensive Färbung innerhalb der Atemwege (große Pfeile) der beta-Galactosidase-behandelten Mäusen nicht aber die Albumin behandelten Mäusen (3A und 3B). _upload/50080/50080fig1.jpg &quot;/&gt; Abbildung 1. Um die Mäuse für die intratracheale Injektion Position war ein Holz-Plattform aufgebaut, dass eine Rampe in einem 45 ° Winkel von der Basis 16. Die Mäuse aus ihren Schneidezähne durch einen Metalldraht, der zwei Metallhaken an der Spitze befestigt ist suspendiert die Rampe. Die Zunge wird vorsichtig mit einer Pinzette und Büroklammer Laryngoskop verwendet wird, um die Atemwege offen positioniert. <img alt="Abbildung 2" src="/files/ftp_upload/50080/50080fig2.jpg" /> Abbildung 2. Nach der Platzierung einer Lichtquelle auf den Hals, kann der Kehlkopf visualisiert werden für intratrachealen Injektion 16. <img alt="Abbildung 3" src="/files/ftp_upload/50080/50080fig3.jpg" /> Abbildung 3. β-Galactosidase Immunofärbungen wurden auf 4 um Lunge von Mäusen intratracheal mit 2 ug injiziert durchgeführtAlbumin (A) oder β-Galactosidase (B). Pfeile zeigen β-Galactosidase-Färbung innerhalb Atemwege. Bilder sind bei 40-facher Vergrößerung. Maßstab bar = 100 um.

Watch this Scientific Journal Video about Protein Transfection of Mouse Lung at JoVE.com

Protein Transfection of Mouse Lung

Transgene Mäuse, oder virale Vektoren verwendet, um Protein-Expression in der Lunge verbessern. Allerdings sind diese Techniken zeitaufwendig, technisch anspruchsvoll und haben Off-Target-Effekte, die Ergebnisse verwechseln kann. Unser Protein Transfektionsprotokoll verwendet eine Lipidbasis Transfektionsreagenz und einer ultrafeinen Feinzerstäuber gleichmäßig liefern aktives Protein zu Lungenzellen.

Protein Transfektion von Maus Lung

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

Nanyang Technological University

Research

JoVE Journal

Biology

9.3K Aufrufe.  St. Luke's Roosevelt Medical Center. Transgene Mäuse, oder virale Vektoren verwendet, um Protein-Expression in der Lunge verbessern. Allerdings sind diese Techniken zeitaufwendig, technisch anspruchsvoll und haben Off-Target-Effekte, die Ergebnisse verwechseln kann. Unser Protein Transfektionsprotokoll verwendet eine Lipidbasis Transfektionsreagenz und einer ultrafeinen Feinzerstäuber gleichmäßig liefern aktives Protein zu Lungenzellen.

Serie: Protein Transfection of Mouse Lung

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Langfristige High-Resolution Intravitalmikroskopie in der Lunge mit einem Vakuum-Stabilisierte Imaging-Fenster

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Forschung

Krebsforschung

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-Transplantation Maus-Modell der myeloproliferativen Neoplasien

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Lentivirale vermittelte Produktion von transgenen Mäusen: eine einfache und hocheffiziente Methode für die direkte Untersuchung der Gründer

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Genetik

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

Orthotopen Transplantation von Phänomen Lungenzellen Adenokarzinom, PD-L1 Ausdruck zu studieren

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

A Novel Surgical Ansatz intratracheale Verabreichung von Wirkstoffen in einem Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Medizin

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Nachweis der Lungentumorprogression bei Mäusen durch Ultraschall-Bildgebung

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

<meta charset="utf8"></head><body>Lentivirale Vektorvorbereitung für die in vivo Genmodulation

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Erforschung des Makrophagen-Immunstoffwechsels durch lentivirale Vektor-vermittelte Genmanipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Lentivirale Vektorpräparation für die effiziente Gen- und microRNA-Modulation von geweberesidenten Makrophagen in der Bauchhöhle in vivo bei Mäusen

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Immunologie und Infektion

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

In-vivo-Mikroinjektion und Elektroporation von Maus-Hoden

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Protein Transfektion von Maus Lung

Zusammenfassung

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In-vivo-Mikroinjektion und Elektroporation von Maus-Hoden