Name: Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice (Video) | JoVE Journal
Uploaded: 2024-02-16T12:30:00.000+00:00
Duration: 6 min 33 s
Description: Learn lentiviral vector preparation for efficient gene and microRNA modulation of peritoneal tissue-resident macrophages in vivo using mice. This method enables stable transgene integration for immunology research.

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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 

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</ol>

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 investigate macrophage function in their biological microenvironment.
It is essential for the success of the protocol to use healthy HEK293T cells. It is the best practice to defrost the cells at least a week prior to the start of this protocol to ensure cell recovery and good numbers. Cells should be seeded in an appropriate volume of medium one day before planned transfection and should reach about 80% confluency on the day of transfection. Under- or over-confluent cell preparations will result in reduced lentivirus yield. We recommend transfecting HEK293T cells with the transfection reagent according to the manufacturer&#39;s instructions for the best results. Essential steps of the transfection include appropriate mixing of the plasmids and reagents and dropwise addition of the mix directly to the HEK293T cell monolayer. Calcium phosphate transfection of HEK293T cells27,28 could be employed in this protocol. However, the user should be aware that the effectiveness of this method can vary, and it can result in diminished transfection efficiency.
Safety precautions should be considered in the protocol from the day of HEK293T cell transfection. These include working in a category II safety cabinet, wearing double gloves when handling contaminated material, and appropriate bleaching of the contaminated material (with decontamination solution, for example, 2,000 ppm bleach solution) for minimum 4 h. Users should refer to their institutional regulations regarding work with category II pathogens and waste.
In this protocol, the lentivirus preparation is first purified using a 0.45 &#181;m filter to remove HEK293T cell debris. The use of smaller filters (0.22 &#181;m) and cellulose ester membranes should be avoided as it will result in the loss of lentivirus particles. It is recommended to use low protein binding polyether sulfone or polyvinylidene fluoride filters29. The second purification step is performed on the single 20% sucrose layer in an ultracentrifuge to remove remaining impurities, which is particularly important for the consequent in vivo administration of the lentivirus preparation. In the institutions where an ultracentrifuge is not available, others30 have described sucrose-based lentivirus purification using a standard laboratory centrifuge. This could be implemented in this protocol as an alternative. Lentivirus preparation is titrated in Jurkat T cells on the expression of the marker signal (e.g., GFP used in this protocol). For constructs without markers, physical lentivirus particles could be evaluated by quantification of HIV-1 p24gag protein by ELISA kit31, flow cytometry analysis of HIV-1 core antigen18, or measurement of the changes to the targeted gene (preferentially using the methods allowing measurement of changes in individual cells, e.g., flow cytometry or microscopy). If titration of the control and lentivirus differ significantly in Jurkat T cells, the volumes used for the in vivo studies can be adjusted to reach the most comparable infection between lentivirus preparations.
The major limitation of the protocol is the observed disappearance of GFP+ resident peritoneal M&#966; within 14 days of i.p lentivirus injection. For long-term studies, mouse lines with stable genetic alteration in a specific cell type or tissues should be considered32. For example, as peritoneal macrophages do not express Foxp3 (a T-Reg protein), we utilized Foxp3-DTR-eGFP mice33 and confirmed that expression of GFP is maintained in peritoneal M&#966; at 21 days post-infection (Figure 3F). However, it is important to note that the lentiviruses contain other foreign components, and this extension in the persistence of infected cells in the Foxp3-DTR-GFP mice may not be permanent. An additional weakness of this method is a low level of inflammation that can be observed in the peritoneal cavity following lentivirus injection, as witnessed by influx of neutrophils at 4 h post injection (Figure 5D), which may mean repeated infections would accelerate the inflammation-associated loss of GFP-expressing resident peritoneal M&#966;. Although, we have not detected type I interferons (IFNs) in the peritoneal cavity, VSV-G pseudotyped lentivirus particles were previously demonstrated to induce some of the IFN-stimulated genes in human M&#966; in the absence of detectable IFNs34. This should be considered when using this protocol for experiments investigating antiviral immune responses. The occurrence of acute and sustained inflammation after i.p injection of lentivirus preparation might indicate contamination of the preparation.
Areas of troubleshooting include: 1) for low lentivirus titer, ensure the health of HEK293T cells and effective transfection (e.g., marker expression, if present, in HEK293T cells). If the infection rate remains low, consider the size of the construct. Constructs with larger inserts or more complex secondary structures can affect the final lentivirus titer and infectivity24. Therefore, each construct should be tested independently and in parallel to its respective control vector; 2) If a high amount of the lentivirus is required, it is recommended to prepare multiple T175 flasks of HEK293T cells and to scale up the production accordingly. To avoid variation of the lentivirus preparations, it is the best practice to merge the final collections prior to aliquoting and storage. For such larger production, plasmids mix (section 2) should be prepared in 50 mL tubes to ensure effective mixing of the components.
Despite its broad tropism, VSV-G pseudotyped lentiviral particles predominantly target tissue macrophages as demonstrated previously for alveolar35, and here18 for pM&#1060; when administered by the respective routes. Alterations to the envelope on lentivirus particles can, in some cases, result in diminished transduction of macrophages in vivo36 and are unnecessary for this protocol. Compared to other viral approaches for in vivo gene manipulation in macrophages (reviewed in37), the use of lentiviral vectors offers stable integration of the transgene in tissue macrophages35, efficient transgene expression, and the largest vector size limit (approximately 8 Kb).
Peritoneal M&#966; play an important role in the prevention, onset, progression, and resolution of various diseases, including abdominal cancers, pancreatitis, and peritonitis38. This protocol describes an effective tool for gene modification in murine pM&#966;, allowing investigation of the biological processes behind these pathologies within a physiologically relevant microenvironment. While lentiviral vectors themselves are becoming a tool of interest for clinical interventions39, due to the origin from the immunodeficiency virus and a stable genome integration, the safety concerns impede their therapeutic implementation. Further understanding of the macrophage responses to lentiviral gene modulation could advance the application of this highly effective tool in a clinical setting.

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><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 &amp;mu;m sterile millex GP filter</td><td>Merck</td><td>SLGS033SS</td><td></td></tr><tr><td>0.45 &amp;mu;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 &amp;mu;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 cm&lt;sup&gt;2&lt;/sup&gt;, 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 &amp;amp; 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 &amp;micro;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)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td>heat inactivated for 30 min at 56 &amp;deg;C and sterile filtered through 0.22 &amp;mu;m filter</td></tr><tr><td>Flow cytometer</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;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&#039;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 &amp;mu;L)</td><td>Fisher Scientific &amp;amp; Starlab</td><td>11963466 &amp;amp; 11943466 &amp;amp; 11973466 &amp;amp; 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-&amp;Delta;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&#039;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, &amp;kappa; 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&#039;s PBS (DPBS) (1x) Mg&lt;sup&gt;++&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt; - 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 &amp;amp; 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

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.

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

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

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.

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

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

This protocol aims to introduce a method to produce and purify mesenchymal stem cell (MSC)-derived extracellular vesicle biomimetics for gene therapy. The nanovesicle is confined with MSC-derived lipid bilayers and encapsulates recombinant AAVs carrying the gene of interest in the lumen. This nanovesicle offers an enhanced vector for in vivo gene delivery.

Development of Mesenchymal Stem Cell Membrane-Enveloped Nanovesicles for Enhanced Gene Delivery

Learn lentiviral vector preparation for efficient gene and microRNA modulation of peritoneal tissue-resident macrophages in vivo using mice. This method enables stable transgene integration for immunology research.

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice (Video) | JoVE Journal

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice

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