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 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

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.

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

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

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 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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.1K Views.  Cardiff University. Vergeleken met andere virale benaderingen voor in vivo genmanipulatie in macrofagen, biedt het gebruik van lentivirale vectoren een stabiele integratie van de transgene weefselmacrofagen, efficiënte transgenexpressie en de limiet voor de grootste vectorgrootte. Bij het in vivo toepassen van lentivirale manipulatie van metabole enzymen, ontdekten we opmerkelijke functionele veranderingen in weefselresidente macrofagen. Daarom onderzoeken we momenteel het immu...

Video: Auteur in de schijnwerpers: onderzoek naar het immunometabolisme van macrofagen door middel van lentivirale vectorgemedieerde genmanipulatie

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

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

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.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Behavior

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

Genetics

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

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

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

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.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

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

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

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

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

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

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

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.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Medicine

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

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

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.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

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

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

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.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

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.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

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

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

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

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.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

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

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