<meta charset="utf8"></head><body>用于体内基因调节的慢病毒载体制备

<ol>
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<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

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.

<meta charset="utf8"></head><body>作者聚焦：通过慢病毒载体介导的基因操作探索巨噬细胞免疫代谢

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

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.

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

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

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

494 次观看.  Cardiff University. 与巨噬细胞体内基因操作的其他病毒方法相比，使用慢病毒载体提供了转基因组织巨噬细胞的稳定整合、高效的转基因表达和最大的载体大小限制。当采用代谢酶的体内慢病毒操作时，我们发现组织驻留巨噬细胞的显着功能变化。因此，我们目前正在研究免疫代谢及其在保持组织特异性功能（包括表观遗传学和免疫功能）中的作用。未来，我们...