小鼠体内siRNA转染CD40沉默和肾免疫染色评价

Miguel  Hueso; Adrián Mallén; Elia  Ripoll; Laura de Ramón; Nuria  Bolaños; Cristian Varela; Jordi  Guiteras; Javier  Checa; Estanislao  Navarro; Josep Maria  Grinyo; Josep Maria Cruzado; Josep Maria  Aran; Joan Torras

doi:10.21769/BioProtoc.4032

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Peer-reviewed

In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining

小鼠体内siRNA转染CD40沉默和肾免疫染色评价

EN Estanislao Navarro

JG Josep Maria Grinyo

JC Josep Maria Cruzado

Josep Maria Aran

JT Joan Torras email

发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4032 浏览次数: 8395

评审: Fereshteh AzediAnonymous reviewer(s)

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Jun 2013

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Abstract

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our group has broad experience in silencing CD40 using RNAi technology, and here we summarize protocols for the systemic administration of a specific anti-CD40 siRNA in different rodents models, in addition to the subsequent quantification of CD40 expression in murine kidneys by immunostaining. The use of RNAi technology with specific siRNAs to silence genes is becoming an essential method to investigate gene functions and is rapidly emerging as a therapeutic tool.

Graphic abstract:

CD40 siRNA mechanism

Keywords: siRNA (siRNA)

CD40 (CD40)

CD40L (CD40L)

Cholesterol-derived oligonucleotide (胆固醇衍生的寡核苷酸)

Kidney (肾脏)

siRNA therapy (siRNA治疗)

Off-target effects (非目标效应)

Background

The co-stimulatory molecule CD40 and its ligand CD40L are one of the best characterized immune checkpoints involved in the pathophysiology of autoimmune and inflammatory diseases, including cancer, Graft-versus-Host-Disease, inflammatory bowel diseases, systemic lupus erythematosus (SLE), rheumatoid arthritis, type 1 diabetes mellitus, allograft rejection, and atherosclerosis (Lutgens et al; 2010; Ripoll et al., 2013; deRamón et al., 2015; Hueso et al., 2016; Hueso et al., 2019; Karnell et al., 2019). CD40 is a 43-50 kDa transmembrane protein belonging to the tumor necrosis factor (TNF) receptor superfamily and is expressed on the surface of many immune cells. The interaction of CD40 with its ligand CD40L (CD154) induces the trimerization of CD40 and stimulates downstream signaling, including the NF-κB pathway that upregulates proinflammatory genes (Elgueta et al., 2009). Thus, inhibition of the CD40-CD40L dyad is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality, such as the administration of small molecule inhibitors of the interaction of CD40 with TRAF6 (Bosmans et al., 2020), liposome-loaded anti-CD40 antisense oligonucleotides (ASO) (Arranz et al., 2013), anti-CD40 siRNAs (Pluvinet et al., 2004; deRamón et al., 2015; Hueso et al., 2016), or monoclonal antibodies (Remer et al., 2017). Currently, there are three clinical trials testing the safety and efficacy of the anti-CD40 monoclonal antibody, CFZ533, to prevent acute rejection in renal (NCT03663335) or liver (NCT03781414) transplant patients and to evaluate its effects on the kidney in patients with lupus nephritis (NCT03610516). Other clinical trials under development use an anti-CD40L antibody (NCT03605927) to prevent acute Graft-Versus-Host-Disease (NCT03605927), or an oncolytic adenoviral vector that expresses an anti-CD40 antibody (NCT03852511) to treat advanced or metastatic tumors. Our group has developed a chemically stabilized, cholesterol-conjugated small inhibitory RNA molecule against murine CD40 and reported the evaluation of its potency, distribution, and durability of effects following systemic administration (Ripoll et al., 2013; deRamón et al., 2015, Hueso et al., 2016, Hueso et al., 2019). Here, we summarize a protocol describing the systemic administration of this specific anti-CD40 siRNA in different mouse models.

Materials and Reagents

Consumables
1. Disposable gloves
2. RNAase-free 1.5 ml polypropylene tubes
3. 96-well plates
4. 0.5 ml polypropylene tubes
5. 50 ml Falcon conical tubes (Corning, catalog number: 45352054)
6. 70 μm nylon cell strainers (BD Biosciences , catalog number: 45352350)
7. Cell culture plates
8. Syringes
9. Towels
10. Sample collection tubes
11. 23-25 G needles
12. 2 ml RNAase-free Eppendorf tubes (Merck KGaA, catalog number: T2795)
13. Poly-L-lysine-coated slides (Merck KGaA, catalog number: P0425)
14. Whatman No. 1 filter paper

Reagents and Kits

Nuclease-free water (DEPC-treated water)
Absolute isopropanol (Merck KGaA, catalog number: I9516)
Tris base (Tris-hydroxymethyl-aminomethane; Bio-Rad Lab, catalog number: 161-0719)
3 M sodium acetate (Merck KGaA, catalog number: S7899)
Bromophenol Blue-Xylene Cyanole Dye Solution (Merck KGaA, catalog number: B3269-5mL)
N,N,N’,N’-tetramethylethylenediamine, TEMED (Bio-Rad, Hercules, catalog number: 161-0800)
Amonium persulfate, APS (Bio-Rad, Hercules, catalog number: 161-0700)
Methanol (Merck KGaA, catalog number: 82762)
Absolute ethanol (Obtained from general chemical providers)
Hydrogen peroxide (Obtained from general chemical providers)
D+ sucrose (AppliChem GmbH, catalog number: 200-334-9)
Ethylenediaminetetraacetic (EDTA, Merck KGaA, catalog number: E6758)
RNase-ZAP (Merck KGaA, catalog number: R2020)
BHT (Butylated hydroxytoluene; Merck KGaA, catalog number: W218405)
FACS lysing solution (Becton Dickinson, catalog number: 349202)
Xylene (mixture of isomers, VWR International, catalog number: 28975.360)
DPX mounting medium (VWR International, catalog number: 360294H)
Jelly from porcine skin (Sigma-Aldrich, catalog number: G1890)
Oil-Red O (Merck KGaA, catalog number: O0625)
RPMI 1640 medium (Biological Industries)
Note: Reagents 1-20 were stored at room temperature.
Opti-MEM (Themo Fisher, catalog number: 51985034)
Fetal bovine serum (Lonza Pharma&Biotech, catalog number: 14-802F)
Albumin ELISA kit (Active Motif)
DEPC (Diethyl Pyrocarbonate; Merck KGaA, catalog number: D5758).
40% acrylamide/Bis 29:1 (Bio-Rad, Hercules, catalog number: 161-0146)
Propidium iodine (StemCell Technologies, catalog number: 75002)
TRIzol (ThermoFisher Scientific, catalog number: 15596026)
DAB substrate (3,3’Diaminobenzidine tetrahydrochloride hydrate, Sigma-Aldrich, catalog number: D5637-5G)
Flow cytometry staining buffer (ThermoFisher Scientific, catalog number: 00-4222)
100 mM Penicilin/Streptomycin (ThermoFisher Scientific, catalog number: 15070-063)
Note: Reagents 21-30 were stored at 4°C.
200 mM L-glutamine (ThermoFisher Scientific, catalog number: 25030-024)
High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, catalog number: 4368814)
PureLinkTMRNA Mini kit (ThermoFisher Scientific, catalog number: 12183020)
5’ RACE system for rapid amplification of cDNA ends kit (ThermoFisher Scientific)
Oligofectamine 2000 (ThermoFisher Scientific, catalog number: 12252011)
GM-CSF (R&D systems, catalog number: 215-GM)
Lipopolysaccharides (LPS) from E. coli Serotype O111:B4 (Sigma-Aldrich, catalog number: L4391)
GeneEraser Luciferase Suppression-Test System (Stratagene/Agilent, catalog number: 240192)
Luciferase Assay System (Promega, catalog number: E1531)
Polyfect transfection reagent (Qiagen, catalog number: 301105)
Serum normal goat (Merck KGaA, catalog number: G9023) or horse 20% (Merck KGaA, catalog number: H0146)
Note: Reagents 31-41 are stored at -20°C.

Taqman gene expression assays (ABI/ThermoFisher Scientific) (Table 1)

Table 1. Commercial TaqMan probes used in this study

Name of the gene tested	Probe code¹	Gene unique identifier²	Position³
CD40	Mm00441891_m1	NM_011611	121
CD40L	Mm00441911_m1	NM_011616	348
IL1b	Mm01336189_m1	NM_008361	55
NLRP3	Mm00840904_m1	NM_145827	499
Apelin (Apln)	Mm00443562_m1	NM_013912	414
C3	Mm01232779_m1	BC029976	517
CD55	Mm00438377_m1	NM_010016	1154
FOXP3	Mm00475165_m1	NM_001199347	1307
IL6	Mm99999064_m1	NM_031168	233
IL10	Mm00439614_m1	NM_010548	232
MCP1 (Ccl2)	Mm00441242_m1	NM_011333	165
RANTES (Ccl5)	Mm01302428_m1	NM_013653	245
TLR3	Mm00628112_m1	NM_126166	280
TLR4	Mm00445273_m1	NM_021297	112
TLR9	Mm00446193_m1	NM_031178	106
AIM2	Mm01295719_m1	NM_001013779	869
18S rRNA	Hs99999901_s1	X03205	604

(1) Commercial code of the Taqman probes used (ThermoFisher Scientific).

(2) Refseq/Genbank unique identifiers of the genes tested.

(3) Base position contained within the probe.

0.1 M citrate buffer, pH 6 (see Recipes), stored at room temperature
0.01 M citrate buffer (see Recipes), stored at room temperature
Oil-Red working solution (see Recipes), stored at room temperature
Phosphate-buffered saline (PBS), pH 7.5 (see Recipes), stored at room temperature
PBS-Triton (PBST) (see Recipes), stored at room temperature
PS buffer (see Recipes), stored at room temperature
Complete culture medium (see Recipes), stored at 4°C
Secondary antibodies (for a 1:200 dilution) (see Recipes), keep at -20°C
Annealing buffer 10× (see Recipes)
DEPC-treated water (see Recipes)
1 M Tris-HCl, pH 7.5 (see Recipes)
12% acrylamide gel (see Recipes)
Non-denaturing gel loading buffer (see Recipes)
4% PFA solution in PBS (see Recipes)
0.3% Oil-Red O stock solution (see Recipes)
Serum normal goat or horse 20% (see Recipes)

Primary antibodies
In this work we used the following specific primary antibodies:
1. Rabbit polyclonal anti-CD40 antibody (C-20, Santa Cruz Biotech, catalog number: sc-975)
2. Rabbit polyclonal anti-CD154 antibody (H215, Santa Cruz Biotech, catalog number: sc-9097)
3. Mouse monoclonal anti-DC-SIGN antibody (1B10, Santa Cruz Biotech, catalog number: sc-23926)
4. Rabbit polyclonal anti-NF-κB p65 antibody (anti-phospho-S536, Abcam, catalog number: Ab86299)
5. Anti-F4/80 antibody (Hycult Biotech, catalog number: HM1066)
6. Rabbit anti-mouse collagen-IV antibody (Chemicon international, catalog number: AB756p)
7. Goat polyclonal anti-human C3c antibody conjugated to FITC (Nordic-MuBio, catalog number: GAHu/C3c/FITC)
  
  Furthermore, the following antibodies from BD Biosciences (San Jose, CA, USA) were used for flow cytometry:
8. Anti-CD11c antibody (clone HL3)
9. Anti-CD11b antibody (clone M1/70)
10. Anti-CD40 antibody (clone HM40-3)
11. Anti-CD80 antibody (clone 16-10A1)
12. Anti-CD86 antibody (clone GL1)
  Note: All primary antibodies were stored at -20°C, unless another temperature was specifically stated by the manufacturer.
Secondary antibodies and histological reagents
The following secondary antibodies were used in this work:
1. Alexa 488-labeled chicken anti-goat (ThermoFisher Scientific, catalog number: AB_2535870)
2. Goat anti-rabbit (ThermoFisher Scientific, catalog number: AB_2633280)
3. Alexa 555-labeled goat anti-mouse (ThermoFisher Scientific, catalog number: AB_2535844)
4. Alexa 546-labeled goat anti-rabbit (ThermoFisher Scientific, catalog number: AB_2633280)
5. Unconjugated goat anti-rat (Novus Biologicals)
6. Biotinylated horse anti-goat (Vector Laboratories, catalog number: BA-9500)
7. FITC-conjugated goat anti-mouse (Merck KGaA, catalog number: F0257)
8. VECTASTAIN Elite ABC HRP kit (Vector Laboratories, catalog number: PK-6100)
9. Avidin/Biotin blocking kit (Vector Laboratories, catalog number: PK-4001-NB)
  Note: All secondary antibodies were stored at 4°C, unless another temperature was specifically stated by the manufacturer.
  
  The following histological reagents were used:
10. Harris hematoxylin solution (Sigma-Aldrich, catalog number: HHS32-1L)
11. Eosin Y solution, alcoholic (Sigma-Aldrich, catalog number: HT110132-1L)
12. Periodic acid (Sigma-Aldrich, catalog number: P0430-25G)
13. Schiff reagent (Sigma-Aldrich, catalog number: 3952016-500ML)
14. PAS staining kit (Sigma-Aldrich, catalog number: 101646)
15. Oil Red-O reagent (Sigma-Aldrich, catalog number: O0625-100G)
16. Tissue Tec OCT inclusion compound (Sakura FinetekEurope)
17. UltraCruz Tm mounting medium (Santa Cruz Biotech, catalog number: sc-24941)
18. DRAQ5 (ThermoFisher Scientific, catalog number: 65-0880-92)
Notes:
1. Used for nuclear counterstaining.
2. All histological reagents were stored at room temperature.
Animals
1. Six-month-old NZB/NZW (F1) female mice (The Jackson Lab, Charles River, Wilmington, MA, USA)
2. Six to eight-week-old male ICR mice (The Jackson Lab, Charles River, Wilmington, MA, USA)
3. Eight-week-old ApoE^-/-female mice (The Jackson Lab, Charles River, Wilmington, MA, USA)
  Note: Animals were housed in a room maintained at a constant temperature and given free access to water and a standard laboratory diet. To accelerate atherosclerosis, ApoE^-/- mice were fed a Western diet that contained 0.2% cholesterol and provided 42% of the energy as fat (TD.88137; Harlan-Tekland, Madison, WI, USA). Animals were euthanized by inhalation of isoflurane. Protocols were approved by the Ethics Committee for Animal Research of UB-Bellvitge, and experiments were performed in accordance with the European legislation on Laboratory Animal Experiments.
siRNA oligonucleotides
In this work, we used a CD40-specific ds-siRNA homologous to both the mouse and rat CD40 mRNA sequence (herein antiCD40-siRNA^Chol) and a scrambled sequence (s/s) ds-siRNA as the control (herein s/s-control siRNA^Chol). Both were modified by conjugating a cholesterol (chol) molecule to the 3’ end of the sense strand by means of a pyrrolidine linker.
The siRNA sequences were as follows:
Anti-CD40 sense strand: 5’-GUGUGUUACGUGCAGUGACUU-3’
Anti-CD40 antisense strand: 5’-GUCACUGCACGUAACACACTG-3’
(s/s), control siRNA sense strand: 5’-ACUACAAGACUCGUGACCAUU-3’
(s/s), control siRNA antisense strand: 5’-UGGUCACGAGUCUUGUAGUUU-3’
To determine the transfection efficiencies and organ distributions of the siRNAs, a cholesterol-conjugated and an unmodified anti-CD40 siRNA were labeled with Cy5.5.
Note: All oligonucleotides were obtained from Microsynth AG (Balgach, Switzerland) and stored at -20°C.
Cell lines
1. The highly transfectable human embryonic kidney 293FT cell line (ThermoFisher Scientific, catalog number: R70007)
2. Primary dendritic cells (obtained from the bone marrow of ICR mice)

Equipment

Cell scrapers (Sarstedt AG & CO, Nümbrecht, GE, catalog number: 83.3950)
Scalpels
Forceps: Straight, serrated-tip forceps; straight or curved, serrated-tip fine forceps; and straight fine-tip forceps
Scissors: Straight, blunt scissors; straight, sharp, fine scissors; and micro-dissecting spring scissors
Automatic pipettes (1-10 μl, 20-200 μl)
Hemocytometer
Water bath
Jasco V-650 spectrophotometer (Easton, MD, USA, used to determine the melting point of siRNA duplexes)
Olympum autoanalyzer AU400 (Hamburg, Germany, used to determine urinary protein and creatinine concentrations)
TaqMan real-time PCR ABI Prism^® 7700 (ThermoFisher Scientific, used for the qPCR experiments)
BD FACS Canto II cytometer (BD Biosciences, used in the flow cytometry experiments)
Zeiss SteREOLumar V12 microscope (Carl Zeiss AG, used for microscopy experiments)
Leica TCS-SL spectral confocal microscope (Leica Camera AG, used for microscopy experiments)
TD-20/20 luminometer (Turner Designs, used for luciferase assays)
T-25 ULTRA-TURRAX^TM homogenizer (IKA^®-Werke GmbH & Co)
NanoDrop 2000c spectrophotometer (ThermoFisher Scientific)
Tabletop centrifuge (Eppendorf Cooled Centrifuge, mode: 5424R)
Cell culture CO₂ incubator (NuAire Autoflow NU 8700)

Software

Leica confocal software (Leica Camera AG, Wetzlar, Germany)
Image analysis software ProgResCapturePro 2.7.7 (JenoptiK AG, Jena, GE)
ImageJ v1.48 (NIH, Bethesda, MD, USA)
ExpressionSuite software v1.0 or later (ABI, ThermoFisher Scientific, Waltham, MA, USA)
SDS software v2.4 (ABI, ThermoFisher Scientific, Waltham, MA, USA)
FACS DIVA software (BD Biosciences, San Jose, CA, USA)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Hueso, M., Mallen, A., Ripoll, E., de Ramon, L., Bolaños, N., Valera, C., Guiteras, J., Checa, J., Navarro, E., Grinyo, J. M., Cruzado, J. M., Aran, J. M. and Torras, J. (2021). In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining. Bio-protocol 11(10): e4032. DOI: 10.21769/BioProtoc.4032.