This research was funded, in whole or in part, by the Wellcome Trust Investigator Award [107964/Z/15/Z]. P.R.T is also supported by the UK Dementia Research Institute. M.A.C is supported by the Biotechnology and Biological Sciences Research Council Discovery Fellowship (BB/T009543/1). For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. L.C.D is a lecturer at Swansea University and an honorary research fellow at Cardiff University. This work is supported by work carried out by Ipseiz et al. 202018.

Lentiviral Vector Preparation for In Vivo Gene Modulation 

<ol>
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The authors have nothing to disclose.

Tissue-resident macrophages perform a range of homeostatic and inflammatory tissue-specific functions1,2 dictated by their physiological environment6,7,8,9. In this protocol, an effective method18 for manipulation of peritoneal resident macrophages in vivo using lentivirus particles was introduced to inv...

Tissue-resident macrophages (M&#966;) are a heterogeneous population of phagocytic immune cells that sense and respond to invading pathogens1,2. In addition, they play an essential role in tissue development, remodeling, and maintaining homeostasis1,3. Many tissue M&#966; derive from yolk sac progenitors during embryogenesis and persist in the tissue throughout the life4,5. The phenotype and functions of these cells are dictated by collaborative and hierarchical interactions of specific transcription factors and the local microenvironment6,7,8,9. A growing understanding of this dependency increases the need for effective in vivo methods for gene manipulation of M&#966; within their physiologically relevant niche.
Lentiviral vectors are a frequently employed tool for the manipulation of nucleic acids in specific cell populations in vivo10,11,12, particularly due to their ability to infect both dividing and non-dividing cells and to stably integrate into host genome13,14. Over the last two decades, lentivirus delivery technology has been optimized, and alternative envelopes and synthetic promoters have been investigated to increase lineage-specific targeting8,15. Owing to its broad cell tropism, vesicular stomatitis virus envelop glycoprotein (VSV-G)16,17 has become the &#34;gold-standard&#34; envelope used in lentivirus technology.
In this protocol18, VSV-G pseudotyped lentiviral particles are employed to demonstrate targeted and effective delivery of short hairpin RNA (shRNA) and microRNA (miR) to mouse peritoneal M&#966; (pM&#966;) in vivo, at steady state19. Transgene expression was driven by the spleen focus forming virus (SFFV) promoter. Productive infection of cells was defined by expression of lentivirus-derived enhanced green fluorescent protein (GFP). Utilization of this approach allowed easy readout for in vivo lentivirus experiments to define the optimal dose and the experimental timeframe. Finally, in vivo lentiviral challenge of mice during thioglycolate-induced inflammation revealed the natural propensity for selective pM&#966; infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x) (Trypsin 500 mg/L or 0.02 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m sterile millex GP filter</td>
			<td>Merck</td>
			<td>SLGS033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m sterile millex GP filter</td>
			<td>Merck</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL U-100 insulin syringe with needle, 0.33 mm x 12.7 mm (29 G)</td>
			<td>BD</td>
			<td>324892</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Litre Sharps Container</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2.4G2 antibody (TruStain FcX anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>AimV medium (research grade), AlbuMax Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>31035025</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking buffer</td>
			<td>prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brewer thioglycolate medium</td>
			<td>Sigma-Aldrich</td>
			<td>B2551</td>
			<td>4% stock solution prepared in water, autoclaved and kept frozen.</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>BD</td>
			<td>550993</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>Biolegend</td>
			<td>117317</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>Biolegend</td>
			<td>117333</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD</td>
			<td>560375</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>Biolegend</td>
			<td>152410</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD226</td>
			<td>Biolegend</td>
			<td>128808</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD3e</td>
			<td>BD</td>
			<td>560527</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD3e</td>
			<td>Biolegend</td>
			<td>152313</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>100412</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD73</td>
			<td>eBioscience</td>
			<td>16-0731-82</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD8a</td>
			<td>eBioscience</td>
			<td>48-0081-82</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Cell culture flask (T175 fask, 175 cm2, 550 mL)</td>
			<td>Greiner Bio One</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, conical bottom tubes 25 mm x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>358126</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuges</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Ultracentrifuge and TC centrifuge</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes (15 mL and 50 mL)</td>
			<td>Greiner Bio One</td>
			<td>11512303 &#38; 11849650</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Greiner Bio One</td>
			<td>123277</td>
			<td>or cryotubes</td>
		</tr>
		<tr>
			<td>DMEM medium (1x) + 4.5g/L D-glucose, 400 &#181;M L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>41965-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma-Aldrich</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Effectene transfection reagent</td>
			<td>Qiagen</td>
			<td>301425</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123123</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123133</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123147</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>FceR1</td>
			<td>eBioscience</td>
			<td>48-5898-80</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td>heat inactivated for 30 min at 56 &#176;C and sterile filtered through 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;Attune NxT</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry (FACS) buffer</td>
			<td>prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent tissue culture microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>EVOS FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>User preference</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco, Life Technologies</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cell line</td>
			<td></td>
			<td></td>
			<td>grown for at least a week prior transfection. Mycoplasma free</td>
		</tr>
		<tr>
			<td>HIV-1 Core antigen</td>
			<td>Beckman Coulter</td>
			<td>6604667</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrex surgical scrub, chlorhexiding gluconate 4% w/v skin cleanser</td>
			<td>Ecolab</td>
			<td>3037170</td>
			<td></td>
		</tr>
		<tr>
			<td>I-A/I-E</td>
			<td>Biolegend</td>
			<td>107625</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>ICAM1</td>
			<td>Becton Dickinson</td>
			<td>554970</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Jurkat T cell line</td>
			<td></td>
			<td></td>
			<td>grown for at least a week prior use. Mycoplasma free</td>
		</tr>
		<tr>
			<td>LIVE/DEAD fixable near-IR dead cell stain kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L34975</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly6G</td>
			<td>Biolegend</td>
			<td>127615</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td>here used C57BL/6 females, aged 8-12 weeks (Charles Rivers), unless specified differently</td>
		</tr>
		<tr>
			<td>Microcapillary pipettes (volume range 0.5-1,000 &#956;L)</td>
			<td>Fisher Scientific &#38; Starlab</td>
			<td>11963466 &#38; 11943466 &#38; 11973466 &#38; S1111-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>Biolegend</td>
			<td>108724</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>prepared to 2% w/v in PBS</td>
		</tr>
		<tr>
			<td>pCMV-&#916;R8.91 packaging plasmid</td>
			<td></td>
			<td>Zuffrey, R., et al. 1997</td>
			<td>encodes Gag-Pol HIV protein driven by cytomegalovirus promoter. Ampicilin resistance.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x, 10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner Bio One</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>pHR&#39;SIN-cPPT-SEW plasmid</td>
			<td></td>
			<td>Rosas, M. et al. 2014</td>
			<td>modified for shRNA and miR expression studies. Encodes EGFP marker downstream SFFV promoter and upstream of the Woodchuck hepatitiv virus enhancer. Ampicilin resistance.</td>
		</tr>
		<tr>
			<td>pMD2.G plasmid</td>
			<td></td>
			<td>Naldini, L. et al. 1996</td>
			<td>encodes vesicular stomatis virus g-glycoprotein (VSV-G) envelope. Ampicilin resistance.</td>
		</tr>
		<tr>
			<td>Rat IgG1, &#954; isotype control</td>
			<td>Becton Dickinson</td>
			<td>550617</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Sigma-Aldrich</td>
			<td>R9759-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood ACK lysis buffer</td>
			<td>prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium (1x) + 400 uM L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>21875-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>S4521</td>
			<td></td>
		</tr>
		<tr>
			<td>SiglecF</td>
			<td>BD</td>
			<td>562681</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite Tablets (bleach, 2,000 ppm)</td>
			<td>Guest Medical</td>
			<td>H8818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 24-well cell culture plate</td>
			<td>Greiner Bio One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Dulbecco&#39;s PBS (DPBS) (1x) Mg++ and Ca2+ - free</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile VWR disposable transfer pipets (23.0 mL, 30 cm)</td>
			<td>VWR</td>
			<td>612-4515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Thermo Fisher Scientific</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>User preference</td>
		</tr>
		<tr>
			<td>Syringes (50 mL and 1 0mL)</td>
			<td>Fisher Scientific</td>
			<td>10084450 &#38; 768160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tim4</td>
			<td>Biolegend</td>
			<td>130007</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>U-bottom 96-well cell culture plate</td>
			<td>Greiner Bio One</td>
			<td>650180</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&amp;#45;Mediated Gene Manipulation

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

Compared to other viral approaches for in vivo gene manipulation in macrophages, the use of lentiviral vectors offers a stable integration of the transgenic tissue macrophages, efficient transgene expression, and the largest vector size limit. When employing in vivo lentiviral manipulation of metabolic enzymes, we discovered notable functional changes in tissue resident macrophages. Therefore, we are currently investigating immuno metabolism and its role in preserving tissue specific functions, including the epigenetics and immune function.In the future, we would like to focus on defining how tissue macrophages affect other local cell populations during homeostasis and in disease models, and how these interactions can be employed for disease prevention and resolution.

When followed fully and correctly, this protocol yields a total of 1.5 mL of high-quality lentivirus stock per single preparation, sufficient for twelve in vivo injections at the optimal volume determined in this study18. The success of the transfection can be evaluated early in the protocol. Healthy and confluent HEK293T cells should display, if present in the plasmids, an easily detectable marker signal (e.g., GFP used in this study) after 48 h post plasmid transfection (<strong class="...

Read this Scientific Article Protocol about Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector-Mediated Gene Manipulation at JoVE.com

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

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

lentiviral-vector-preparation-for-efficient-gene-microrna-modulation

Research

JoVE Journal

Immunology and Infection

Transfection of HEK293T Cells Using Non&#45;Liposomal Lipid Transfection Reagent for Lentiviral Production

To begin, add the lentiviral components in a 15-milliliter tube, and make the volume to 600 microliters with EC buffer. Add 36 microliters of enhancer solution, and using a one-milliliter pipette, mix repeatedly, drawing up and releasing a portion of the liquid back into the solution. After five minutes, add 120 microliters of transfection reagent and mix approximately 20 times, as demonstrated previously.Leave the tube at room temperature for 10 minutes. Then add 5.2 milliliters of cDMEM to the tube. Using a disposable transfer pipette, dropwise add the transfection mixture onto the HEK293T cells monolayer cultured in a T175 flask.Distribute the plasmid evenly through a gentle side-to-side rocking motion of the flask. Incubate the culture at 37 degrees Celsius, and 5%carbon dioxide for 48 hours. After incubation under a fluorescent microscope, confirm the effective transfection of HEK293T cells by observing GFP fluorescence.Now tilt the flask, and using a 25-milliliter serological stripette, collect the medium without disturbing the cell monolayer. Transfer the medium to a pre-labeled 50-milliliter tube and close the lid. Add 25 milliliters of warm cDMEM along the walls of the flask without disturbing the monolayer, and return the flask to the incubator.After 24 hours, collect the medium, add the decontamination solution into the flask, and place it horizontally for the next 24 hours.

Sucrose Density Gradient Based Purification of Lentivirus Particles for Gene Delivery 

To begin, remove the plunger from a 50-milliliter syringe, and insert a low protein binding polyethersulfone or a polyvinylidene fluoride 0.45-micrometer sterile filter into the syringe end. Using a 25-milliliter serological Stripette, add the lentivirus transfected HEK293T cell suspension into the syringe and gently return the plunger. Push the plunger slowly and collect the filtrate in a new 50-milliliter tube.Once done, remove the filter from the syringe and dispose of it in the decontamination solution. Add three milliliters of a 20%sucrose solution into the bottom of the ultracentrifuge tube. Next, set the PIPETBOY to the lowest speed.Hold the ultracentrifuge tube at approximately 45 degrees angle and slowly add the filtered lentivirus-containing medium onto the tube wall. Observe the clear separation of layers from the tube. If the total tube volume is less than 29 milliliters, add fresh cDMEM to avoid the tube collapsing during the spin.Wipe the ultracentrifuge bucket with 70%alcohol and put the tube adapter in the bottom of the bucket. Then, place the ultracentrifuge tube into the bucket. Close the bucket and place it into the rack.Pre-cool the ultracentrifuge to four degrees Celsius. Carefully place the bucket into the rotor and turn on the vacuum. Adjust the acceleration and deceleration rate to the lowest setting and centrifuge at 26, 000 RPM for 90 minutes at four degrees Celsius.After centrifugation, turn off the vacuum and carefully remove the rotor without disturbing the samples. Observe the ultracentrifuge to confirm there are no spills and leaks from the buckets. Pour the content of an ultracentrifuge tube in one smooth motion into the waste container.Invert the tube onto a double-layer tissue paper for 10 minutes. Meanwhile, wipe the insides of the bucket with 70%ethanol-soaked tissue paper and air dry it upside down. Using tissue paper, carefully dry any remaining liquid from the rim of the ultracentrifuge tube.Resuspend the pellet in one milliliter of appropriate serum-free media and leave it for 15 minutes. Using a one-milliliter pipette, gently mix the lentivirus preparations and aliquot them into 1.5-milliliter screw-top tubes. Store the lentiviral preparations at minus 80 degrees Celsius.Successful lentivirus preparation achieved an infection rate of over 95%with a five-microliter dose. The mean fluorescent intensity of infected cells increases with higher doses. The intraperitoneal injection of 100 microliters of lentivirus preparation yielded the highest percentage and intensity of the GFP signal in mice peritoneal cells.Time course experiments with 100-microliter lentivirus injection revealed a significant percentage of GFP-expressing resident cells at days three and seven, followed by the disappearance of the infected population at day 14.

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

Lentiviral Vector Preparation for In Vivo Gene Modulation

Özet