This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 82041045 and 82173764), the major project of Study on Pathogenesis and Epidemic Prevention Technology System (2021YFC2302500) by the Ministry of Science and Technology of China, the Chongqing Talents: Exceptional Young Talents Project (CQYC202005027), and the Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0136). The authors are grateful to Dr. Xiaoyan Ding for measuring the hydrodynamic diameter (nm) and polydispersity index (PDI).

1. In vitro transcription of chemically modified mRNA
NOTE: It is required to use nuclease-free tubes, reagents, glassware, pipette tips, etc., because RNases are ubiquitous in the environment, such as laboratory solutions, instrument surfaces, hair, skin, dust, etc. Clean the bench surfaces and pipettes thoroughly before use, and wear gloves to avoid RNase contamination.
<ol>
	<li>Perform linearization of the DNA template.
	<ol>
		<li>Synthesize the open reading frame (ORF) of Metridia luciferase (MetLuc) flanked by a T7 polymerase promoter, 5&#39; untranslated region (UTR), 3&#39;UTR, and poly (A) tails and clone it into the PUC57 vector, which is expressed in bacteria (see Table of Materials). 
		NOTE: The coding sequence of the DNA template is provided in Supplementary File 1.</li>
		<li>Culture bacteria, lyse the bacteria, and purify the plasmid DNA by the corresponding column (see plasmid DNA extraction kit&#160;in the Table of Materials).</li>
		<li>Perform a single 50 &#181;L reaction that contains 2 &#181;L of BamHI, 2 &#181;L of KpnI (see Table of Materials), and 2 &#181;g of plasmid DNA at 37 &#176;C for 1 h, and achieve linearization of the DNA template.</li>
	</ol>
	</li>
	<li>Perform in vitro transcription to generate uncapped-IVT mRNA.
	<ol>
		<li>Perform mRNA synthesis by a single 20 &#181;L reaction using a T7 transcription kit and pseudouridine (see Table of Materials): 10 &#181;L (0.8-1 &#181;g) of linearized DNA template, 1.5 &#181;L (150 mM) of ATP, pseudo-UTP, GTP, and CTP, 2 &#181;L of 10x transcription buffer, 1 &#181;L of T7 enzyme, and 1 &#181;L of nuclease-free water (NF-water). Mix the abovementioned components thoroughly and incubate at 37 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Remove the template DNA.
	<ol>
		<li>Add 1 &#181;L (1 U) of DNase (RNase-free) after the transcription process and incubate at 37 &#176;C for 15 min.</li>
	</ol>
	</li>
	<li>Perform uncapped-IVT mRNA purification using lithium chloride precipitation.
	<ol>
		<li>Purify the uncapped-IVT mRNA using lithium chloride (see Table of Materials) with a working concentration of 10 mM. Add 50 &#181;L of 10 mM lithium chloride to 20 &#181;L of uncapped-IVT mRNA solution.</li>
		<li>Mix the complete volume thoroughly and chill at &#8722;20 &#176;C for 1 h. Collect the uncapped-IVT mRNA for 12 min at 12,000 x g, which routinely produces 80-120 &#181;g of uncapped-IVT mRNA from each single 20 &#181;L reaction.</li>
	</ol>
	</li>
	<li>Perform IVT mRNA capping.
	<ol>
		<li>Perform the IVT mRNA capping using the cap 1 capping system (see Table of Materials). Briefly, remove the secondary structure of 50 &#181;g of uncapped-IVT mRNA by heating at 65 &#176;C for 10 min, then link the 5&#39; end of uncapped-IVT mRNA with cap 1, which is prepared by modifying the m7G cap structure using 2.5 &#181;L of s-adenosylmethionine (SAM) (20 mM) and 4 &#181;L of 2&#39;-O-methyltransferase (100 U) in a 100 &#181;L reaction at 37 &#176;C for 30-60 min.</li>
		<li>Purify 100 &#181;L of the capped IVT mRNA using 250 &#181;L of 10 mM&#160;lithium chloride and dilute in 50 &#181;L of NF-water.</li>
	</ol>
	</li>
	<li>Determine the purity and molecular size of capped-IVT mRNA.
	<ol>
		<li>Measure the concentration of capped-IVT mRNA with a UV-visible spectrophotometer. Using an RNA marker (see Table of Materials), analyze the molecular size of capped-IVT mRNA in a 1% formaldehyde denaturing agarose gel containing 18% formaldehyde (voltage is 120 V). Ensure the uncapped-IVT mRNA and capped-IVT mRNA are stored at &#8722;80 &#176;C. 
		&#8203;NOTE: The IVT mRNA was considered to have good purity in an A260/A280 ratio of 1.8-2.1 and an A260/A230 ratio of 2.0 or slightly higher.</li>
	</ol>
	</li>
</ol>
2. Generation of IVT mRNA/PP-sNp
<ol>
	<li>Prepare poloxamine 704 (T704) stock solution by solubilizing the T704 (see Table of Materials) in NF-water to obtain a 10 mg/mL stock solution. Store the prepared solution at 4 &#176;C. 
	NOTE: T704 contains an X-shaped structure made of an ethylenediamine central group bonded to four chains of poly(propylene oxide) (PPO) blocks and poly(ethylene oxide) (PEO) blocks24. The molecular weight (Mw) of T704 is 5500.</li>
	<li>Prepare the synthetic peptide (sPep, see Table of Materials) stock solution by solubilizing the sPep (sequence: KETWWETWWTEWWTEWKKKKRRRRRKKKKGACSE 
	RSMNFCG) in NF-water to obtain a 2 mg/mL stock solution and store at 4 &#176;C.</li>
	<li>Prepare the IVT mRNA solution by thawing the IVT mRNA (step 1) on ice and centrifuging shortly for 3 s at 300 x g at room temperature before opening the tube. Dilute the IVT mRNA solution to 0.04 &#181;g/&#181;L with NF-water. 
	NOTE: It is recommended to work in a biosafety cabinet whenever possible with the IVT mRNA.</li>
	<li>Prepare T704 and sPep mix solution by diluting the sPep solution to 0.555 &#181;g/&#181;L and the T704 solution to 8 &#181;g/&#181;L with NF-water. Incubate the mixed solution for 15 min at room temperature prior to further use. 
	NOTE: Calculate the required sPep component based on the desired N/P ratio. The N/P ratio is the total number of nitrogen residues (N) within the sPep to the total number of negatively charged phosphate groups (P) within the IVT mRNA. Calculate the required T704 based on the weight/weight (w/w) ratio between T704 and IVT mRNA. It is necessary that the N/P ratio is 5 and the w/w ratio is 100.</li>
	<li>Prepare the IVT mRNA/PP-sNp formulation following the steps below.
	<ol>
		<li>Draw the IVT mRNA solution (step 3) into a 1 mL syringe, ensuring no air gaps or bubbles in the syringe tip. Load the syringe into one side of the cartridge next to the rotating block.</li>
		<li>Fill a 1 mL syringe with the T704 and sPep mix solution (step 4). Remove any bubbles or air gaps at the syringe tip and put the syringe into the other inlet of the pump (see Table of Materials).</li>
		<li>Set the pump with a flow ratio of 1:1 and a total flow rate of 4-10 mL/min. 
		NOTE: A total flow rate of 6 mL/min is optimal in the studies presented here.</li>
		<li>Place a 10 mL RNase-free conical tube (see Table of Materials) to collect the mixed IVT-mRNA/PP-sNp solution at the end of the flow path of the mixing device.</li>
		<li>Run the pump to start the mixing, ensuring the parameters are input correctly. After the pump is finished running for 6 s, collect the IVT-mRNA/PP-sNp sample from the conical tube. 
		NOTE: PP-sNp&#160;were generated using a microfluidic mixer (Supplementary Figure 1). The device comprises a constant flow pump, link device, chip, and fixed device. In the mixing process, the constant flow pump connected to the chip delivers liquid to the chip according to the preset flow rate. The connected constant flow pump can be connected to multiple input channels of the chip with a single output channel. The components of the device, including the chip, were obtained from commercial sources and rationally assembled (see Table of Materials). The parameters, such as flow rate and volume of the IVT mRNA solution and T704-sPep mixed solution, may differ from those displayed in the current protocol if some different setup is used and must be optimized accordingly.</li>
	</ol>
	</li>
</ol>
3. Measurement of the hydrodynamic diameter and polydispersity of IVT-mRNA/PP-sNp
<ol>
	<li>Dilute an aliquot of the IVT mRNA/PP-sNp solution (step 2) with NF-water to obtain a final volume of 1 mL.</li>
	<li>Measure the hydrodynamic size and polydispersity index (PDI) using a semi-micro cuvette25. Add the IVT mRNA/PP-sNp solution into the cuvette and place it into the particle size meter (see Table of Materials). Set up a standard operating procedure and click on Start to begin the data acquisition.</li>
</ol>
4. Preparation of the cells for transfection
<ol>
	<li>Plate human bronchial epithelial cells (16HBE) and a dendritic cell line (DC2.4) in 96-well-plates at a density of 3.5 x 104 cells/well 24 h prior to transfection. Grow the cells in a culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. 
	NOTE: The cells were obtained from a commercial source (see Table of Materials).</li>
	<li>Incubate the cells in an incubator (37 &#176;C and 5% CO2 atmosphere) for 24 h to ensure the cells are 60%-80% confluent before the transfection.</li>
</ol>
5. Transfection of the cultured cells
<ol>
	<li>After removing the growth medium, wash the plated cells with 0.2 mL/well 1x PBS.</li>
	<li>Add 170 &#181;L of serum-free culture medium to each well containing the plated cell cultures.</li>
	<li>Add the IVT mRNA/PP-sNp formulation containing 0.4 &#181;g of MetLuc mRNA (prepared in step 2) dropwise with an amount of 30 &#181;L/well.</li>
	<li>Incubate the cells with the IVT mRNA/PP-sNp formulation at 37 &#176;C in a humidified 5% CO2-enriched atmosphere for 4 h.</li>
	<li>Replace the transfection medium with 0.2 mL of fresh culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% (v/v) penicillin/streptomycin. 
	NOTE: The fetal bovine serum was heated at 56 &#176;C for 30 min for inactivation.</li>
	<li>Incubate the transfected cells at 37 &#176;C and in a 5% CO2-enriched atmosphere for 24 h and collect the supernatants to be detected from each well.</li>
</ol>
6. Analysis of cell transfection efficacy using Metridia luciferase (MetLuc) assay
<ol>
	<li>Assay the supernatant from each well for MetLuc activity using coelenterazine substrate (see Table of Materials) following the steps below.
	<ol>
		<li>Prepare a fresh assay solution by adding 1x PBS to the coelenterazine substrate (the concentration of coelenterazine is 15 mM).</li>
		<li>Vortex the coelenterazine solution for 10 s for thorough mixing.</li>
		<li>Add 50 &#181;L of supernatant (collected in step 5) to a 96-well plate.</li>
		<li>Set up the microplate reader (see Table of Materials) with a 1,000 ms reading time before adding 30 &#181;L of coelenterazine solution (15 mM) to each well manually or by automated injection.</li>
		<li>Click on Start to measure the luminescence signal immediately after adding the coelenterazine solution to the supernatant. 
		NOTE: The luminescence signal is measured using a microplate reader, and its activity is expressed in relative light units. The values obtained from PBS-transfected wells will be used as blank controls.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The protocol described here not only allows the cost-effective and rapid production of IVT mRNA vaccine formulations with defined properties, but it also offers the possibility to customize the PP-sNp formulation according to specific therapeutic purposes, such as gene therapy. In order to ensure the successful generation of IVT mRNA/PP-sNp, it is suggested to pay extra attention to some critical steps. When working with mRNA, always remember that RNase-free conditions should be maintained throughout the process because ...

Vaccination has been heralded as one of the most efficient medical interventions for reducing the morbidity and mortality caused by infectious diseases1. The importance of vaccines has been demonstrated since the outbreak of coronavirus disease 2019 (COVID-19). As opposed to the traditional concept of injecting inactivated or live-attenuated pathogens, state-of-the-art vaccine approaches, such as nucleic acid-based vaccines, concentrate on preserving the immune-stimulatory properties of the target pathogens while avoiding the potential safety issues associated with the conventional whole-microbial virus- or in bacteria-based vaccines. Both DNA- and RNA (i.e., in vitro transcribed messenger RNA, IVT mRNA)-based vaccines exhibit prophylactic to therapeutic potential against a variety of diseases, including infectious diseases and cancers2,3. In principle, the potential of nucleic acid-based vaccines relates to their production, efficacy, and safety4. These vaccines can be manufactured in a cell-free manner to allow cost-effective, scalable, and rapid production.
A single nucleic acid-based vaccine can encode multiple antigens, enabling the target of numerous viral variants or bacteria with a reduced number of inoculations and strengthening the immune response against resilient pathogens5,6.&#160;Besides, nucleic acid-based vaccines could mimic the natural invasion process of virus or bacterial infection, bringing both B cell- and T cell-mediated immune responses. Unlike some virus- or in DNA-based vaccines, IVT mRNA-based vaccines offer a huge advantage in terms of safety. They can rapidly express the desired antigen in the cytosol and are not integrated into the host genome, obviating concerns about insertional mutagenesis7. IVT-mRNA is automatically degraded after successful translation, so its protein expression kinetics can be easily controlled8,9. Catalyzed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, efforts from companies/institutions worldwide have enabled the release to the market of many types of vaccines. IVT mRNA-based vaccine technology shows great potential and, for the first time, has demonstrated its previously anticipated success, owing to its rapid design and flexible capacity to adapt to any target antigens within several months. The success of IVT mRNA vaccines against COVID-19 in clinical applications not only opened up a new era of IVT mRNA vaccine research and development but also accumulated valuable experience for the rapid development of effective vaccines for dealing with outbreaks of infectious diseases10,11.
Despite the promising potential of IVT mRNA vaccines, the efficient intracellular delivery of IVT mRNA to the site of action (i.e., cytoplasm) continues to pose a major hurdle12, especially for those administered via the airways4. IVT mRNA is inherently an unstable molecule with an extremely short half-life (~7 h)13, which renders IVT mRNA highly prone to degradation by the ubiquitous RNase14. The lymphocytes of the innate immune system tend to engulf the recognized IVT mRNA in cases of in vivo application. Moreover, the high negative charge density and large molecular weight (1 x 104-1 x 106 Da) of IVT mRNA impair its effective permeation across the anionic lipid bilayer of cellular membranes15. Therefore, a delivery system with certain bio-functional materials is required to inhibit the degradation of the IVT mRNA molecules and facilitate cellular uptake16.
Apart from a few exceptional cases in which naked IVT mRNA was directly utilized for in vivo investigations, various delivery systems are used to carry IVT mRNA to the therapeutic site of action17,18. Previous studies have revealed that only a few IVT mRNAs are detected in cytosol without the assistance of a delivery system19. Numerous strategies have been developed to improve RNA delivery with continuous efforts in the field, ranging from protamine condensation to lipid encapsulation20. Lipid nanoparticles (LNPs) are the most clinically advanced among the mRNA delivery vehicles, as proved by the fact that all the approved mRNA COVID-19 vaccines for clinical use employ LNP-based delivery systems21. However, LNPs cannot mediate effective mRNA transfection when the formulations are delivered via the respiratory route22, which remarkably limits the application of these formulations in inducing mucosal immune responses or addressing pulmonary-related diseases such as cystic fibrosis or &#945;1-antitrypsin deficiency. Therefore, developing a novel delivery system to facilitate the efficient delivery and transfection of IVT mRNA in airway-related cells is required to solve this unmet need.
It has been confirmed that the peptide-poloxamine self-assembled nanoparticle (PP-sNp) delivery system can mediate the efficient transfection of nucleic acids in the respiratory tract of mice23. The PP-sNp adopts a multifunctional modular design approach, which can integrate different functional modules into the nanoparticles for rapid screening and optimization23. The synthetic peptides and electrically neutral amphiphilic block copolymers (poloxamine) within the PP-sNp can spontaneously interact with IVT mRNA to generate uniformly distributed nanoparticles with a compact structure and smooth surface23. PP-sNp&#160;can improve the gene transfection effect of IVT mRNA molecules in cultured cells and the respiratory tract of mice23. The present study describes a protocol for generating PP-sNp&#160;containing IVT mRNA that encodes Metridia luciferase (MetLuc-mRNA) (Figure 1). Controlled and rapid mixing via a microfluidic mixing device, which employs the staggered herringbone mixing design, is utilized in this protocol. The procedure is easy to execute and allows the generation of PP-sNp&#160;with more uniform sizes. The general goal of PP-sNp production using the microfluidic mixer is to create PP-sNp&#160;for mRNA complexation in a well-controlled manner, thus allowing efficient and reproducible cell transfection in vitro. The present protocol describes the preparation, assembly, and characterization of PP-sNp&#160;containing MetLuc-mRNA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BamHI</td>
			<td>Takara</td>
			<td>1010</td>
			<td></td>
		</tr>
		<tr>
			<td>cap 1 capping system</td>
			<td>Jinan</td>
			<td>M082</td>
			<td></td>
		</tr>
		<tr>
			<td>Dendritic cell-line</td>
			<td>Sigma</td>
			<td>SCC142</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA sequence</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human bronchial epithelial cells</td>
			<td>Sigma</td>
			<td>SCC150</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI</td>
			<td>Takara</td>
			<td>1068</td>
			<td></td>
		</tr>
		<tr>
			<td>LP</td>
			<td>Beyotime</td>
			<td>C0533</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>APEXBio</td>
			<td>B6083</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern Zetasizer Nano ZS90</td>
			<td>Malvern</td>
			<td>NB007605</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic chip</td>
			<td>ZHONGXINQIHENG</td>
			<td>Standard PDMS chip</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate readers</td>
			<td>ThermoFisher</td>
			<td>Varioskan lux</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>ThermoFisher</td>
			<td>ND-ONE-W (A30221)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudouridine</td>
			<td>APE&#215;Bio</td>
			<td>B7972</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Quanti-Luc</td>
			<td>InvivoGen</td>
			<td>Rep-qlc2</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboRuler High Range RNA Ladder</td>
			<td>ThermoFisher</td>
			<td>SM1821</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free conical tube</td>
			<td>Biosharp</td>
			<td>BS-100-M</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>ThermoFisher</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Chemyx</td>
			<td>Fusion 101</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 transcription Kit</td>
			<td>Jinan</td>
			<td>E131</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient transfection of in vitro transcribed mrna in cultured cells using peptide-poloxamine nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp&#160;encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp&#160;is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp&#160;is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp&#160;nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

The recombinant plasmid was digested to produce the linearized DNA template (Figure 2A). Using the protocol described, The T7 in vitro transcription kit can produce up to 80-120 &#181;g of uncapped MetLuc-mRNA per 20 &#181;L reaction and 50-60 &#181;g of capped MetLuc-mRNA per 100 &#181;L reaction. When analyzed with electrophoresis, intact MetLuc-mRNA with high quality should show a single and clear band, as displayed in Figure 2B. Contaminants introdu...

Watch this Scientific Journal Video about Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles at JoVE.com

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

efficient-transfection-vitro-transcribed-mrna-cultured-cells-using

Research

JoVE Journal

Immunology and Infection

3.1K Views.  Third Military Medical University. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

Video: Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...