eoe sub articles

EoE Sub Articles

1. Transduction of murine and human primary NK cells with lentivirus
<ol>
	<li>Suspend mouse or human primary NK cells in a 24-well plate at 0.5 &#215; 105/mL of medium in the presence of green fluorescent protein (GFP) lentivirus supernatant at 5, 10, and 20 multiplicity of infection (MOI) and in the presence of Pb (8 &#181;g/mL), PS (8 &#181;g/mL) or dextran (8 &#181;g/mL).</li>
	<li>Centrifuge the plates at 1,000 &#215; g for 60 min.</li>
	<li>Without decanting the supernatant, culture the cells overnight (16-18 h) in a 37 &#176;C incubator infused with 5.2% CO2.</li>
	<li>Wash with 10 mL of PBS and resuspend in 2 mL of complete Roswell Park Memorial Institute (RPMI) 1640 culture medium in the presence of interleukin 2 (IL-2, 300 U/mL).</li>
	<li>Harvest the transduced NK cells on day 7 by gently tapping the flasks.
	<ol>
		<li>Activate the harvested cells with titrated concentrations of plate-bound anti-natural killer group 2D (NKG2D) (A10) mAb by coating 96-well, highly protein-absorbent polystyrene plates with 2.5 &#181;g/mL concentrations of anti-NKG2D mAb overnight.</li>
		<li>Wash each well with 100 &#181;L of phosphate-buffered saline (PBS) three times before the addition of primary NK cells.</li>
	</ol>
	</li>
	<li>Collect culture supernatants using a multi-channel pipette between 16 and 18 h post-activation to quantify cytokines such as interferon-gamma (IFN-&#947;). Generate standard curves using the recombinant cytokines provided with enzyme-linked immunosorbent assay (ELISA) kits.</li>
	<li>Test the NK cell viability using annexin-V/7-amino-actinomycin D (7-AAD) staining and determine the percent of necrotic cells among the transformed NK cells using a flow cytometer.
	<ol>
		<li>To perform this assay, harvest the murine and human NK cells four days after transduction, wash two times with 10 mL of cold PBS at 500 &#215; g for 5 min each, and incubate with an Annexin-V (PE)/7-AAD kit.</li>
	</ol>
	</li>
	<li>Use appropriate flow cytometry software to analyze the data. Select the live-cell population and analyze the expression of GFP (fluorescein isothiocyanate [FITC] channel) as a measure of viral transduction.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>90-64-91-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene (Pb)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Protamine sulfate (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>p3369</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (IFN-&#947; )</td>
			<td>eBioscience</td>
			<td>14-7311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium lodide staining solution</td>
			<td>BD</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>NDC 57319-559-05</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kits</td>
			<td>Ebioscience</td>
			<td>00-4201-56</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhancing viral transduction in natural killer cells using a cationic polymer

Source: Nanbakhsh, A., et al. <a href="https://app.jove.com/v/55063">Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an assay for cationic polymer-mediated viral transduction in natural killer (NK) cells. The cells are incubated with transgenic lentivirus vectors encoding a fluorescent protein. The addition of a cationic polymer enables the virus to attach to the cells and integrate its RNA into the host genome, expressing the fluorescent protein. The fluorescence is quantified to assess the polymer-induced enhancement of transduction.

Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer

1. Transduction of murine and human primary NK cells with lentivirus

	Suspend mouse or human primary NK cells in a 24-well plate at 0.5 &#215; 105/mL of ...

Take natural killer, or NK, cells. Add lentivirus vectors comprising viral enzymes and engineered RNA molecules that encode a fluorescent protein.
Add cationic polymers and sediment the cells and viruses.
The polymers neutralize the negative charges on both the virus and the cell, facilitating membrane fusion.
The RNA is reverse-transcribed into DNA and integrated into the host genome, enabling fluorescent protein expression.
Add media supplemented with the cytokine interleukin-2, or IL-2.
IL-2 triggers intracellular signaling that promotes NK cell survival and proliferation while preserving transgene expression.
Transfer the mixture to a well coated with antibodies that bind to specific immunoreceptors on NK cells, triggering them to release antiviral cytokines.
Quantify the released cytokines to assess the polymer&#39;s impact on cytokine production.
Add fluorescent dyes to label the DNA of necrotic cells.
Using flow cytometry, identify live cells and quantify the fluorescent protein&#39;s signal to measure the efficiency of polymer-induced transduction.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

94 vues. Source : Nanbakhsh, A., et al. Le dextran améliore l’efficacité de la transduction lentivirale des cellules NK primaires murines et humaines. J. Vis. Exp. (2018). Cette vidéo présente un test de transduction virale médiée par un polymère cationique dans des cellules tueuses naturelles (NK). Les cellules sont incubées avec des vecteurs de lentivirus transgéniques codant pour une protéine fluorescente. L’ajout d’un polymère cationique permet au virus de se fixer aux cellules et d...

Vidéo: Amélioration de la transduction virale dans les cellules tueuses naturelles à l’aide d’un polymère cationique - Concept

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Utilisation de CRISPR/Cas9 pour éliminer gm-CSF dans les cellules CAR-T

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Recherche

JoVE Journal

Recherche en cancérologie

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Transduction transitoire des formes strobilées d’Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Génétique

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Oeufs fécondés de livraison assistée par laser des gènes gène aux souris

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer

Transcription

Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer