模式生物小鼠口腔微生物组鉴定

Loreto Abusleme; Bo-Young Hong; Anilei Hoare; Joanne E. Konkel; Patricia I. Diaz; Niki M. Moutsopoulos

doi:10.21769/BioProtoc.2655

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Peer-reviewed

Oral Microbiome Characterization in Murine Models

模式生物小鼠口腔微生物组鉴定

PD Patricia I. Diaz email

NM Niki M. Moutsopoulos email

发布: 2017年12月20日第7卷第24期 DOI: 10.21769/BioProtoc.2655 浏览次数: 9752

评审: Ivan ZanoniYang FuAnonymous reviewer(s)

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Cover of Immunity, featuring study using the protocol.

Jan 2017

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Abstract

The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.

Keywords: Oral microbiome (口腔微生物组)

16S rRNA gene sequencing (16S rRNA基因测序)

Microbiome sequencing (微生物组测序)

Murine oral microbiota (小鼠口腔微生物群)

Gingival microbiome (牙龈微生物组)

Mouse oral sampling (小鼠口腔取样)

Background

The microbiome plays critical roles in modulating tissue-specific immune responses particularly at barrier sites (Belkaid and Harrison, 2017). In these barrier environments, such as the gastrointestinal tract and skin, select commensals are shown to be able to drive the development of specific immune cell populations (Ivanov et al., 2009; Naik et al., 2012). Our work has recently started to explore the influence of the oral microbiome in tailoring tissue immunity, particularly at the gingiva, a vulnerable oral barrier site (Abusleme and Moutsopoulos, 2016; Dutzan et al., 2017).

In humans, it is well recognized that the oral cavity harbors a diverse and rich microbiome (Human Microbiome Project, 2012). Alterations in oral microbial communities have been associated with the common oral disease, periodontitis, an inflammatory condition that affects the gingival tissues and results in tissue damage (Griffen et al., 2012; Abusleme et al., 2013; Moutsopoulos et al., 2015). To date, animal models have been instrumental in addressing the role of the microbiome in various physiological and pathological conditions (Turnbaugh et al., 2006; Kostic et al., 2013). However, studies of host-microbiome interactions have been increasingly challenging in the oral murine setting. To facilitate oral microbiome studies in murine models, we have developed protocols for sampling of oral microbial communities, processing of low biomass murine oral microbiome samples, 16S rRNA gene sequencing and analysis of relevant data.

Materials and Reagents

Oral and gingival sample collection
1. Ultra-Fine polystyrene swab (Puritan Medical Products, catalog number: 25-800 1PD 50 )
2. Safe-lock Biopur 1.5 ml Individually wrapped tubes (Eppendorf, catalog number: 022600028 )
3. KIMWIPES delicate task wipers (KCWW, Kimberly-Clark Professional, catalog number: 34120 )
4. Sterile scalpel handle #3 (Fine Science Tools, catalog number: 10003-12 )
5. Scalpel blades #10 (Fine Science Tools, catalog number: 10010-00 )
6. 70% ethanol
7. TE buffer (Epicentre, catalog number: MTE0970 )
8. RNase AWAY decontamination reagent (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10328011 )
9. Ketamine 100 mg/ml (Zetamine CIII, VetOne)
10. Xylazine 20 mg/ml (AnaSed, Lloyd laboratories)
DNA isolation
1. Safe-lock Biopur 1.5 ml Individually wrapped tubes (Eppendorf, catalog number: 022600028 )
2. DNA IQ spin baskets (Promega, catalog number: V1225 )
3. DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
4. Ready-Lyse lysozyme solution (Epicentre, catalog number: R1810M )
5. Ethanol for molecular biology (Sigma-Aldrich, catalog number: E7023 )
6. RNase AWAY decontamination reagent (Thermo Fisher Scientific, catalog number: 10328011 )
16S rRNA gene library amplification and visualization
1. DNA low-bind tube 1.5 ml (Eppendorf, catalog number: 0030108051 )
2. DNA low-bind tube 2.0 ml (Eppendorf, catalog number: 0030108078 )
3. PCR tubes 0.2 ml without caps (Bio-Rad Laboratories, catalog number: TLS0801 )
4. PCR tube 8-Cap strips (Bio-Rad Laboratories, catalog number: TCS0803 )
5. Platinum Taq DNA polymerase high fidelity (Thermo Fisher Scientific, catalog number: 11304011 )
6. dNTP mix (10 mM each) (Thermo Fisher Scientific, catalog number: R0191 )
7. PCR water (QIAGEN, catalog number: 17000-10 )
8. Forward primer 8F 5’-AGAGTTTGATCMTGGCTCAG-3’ (Custom order–IDT)*
9. Reverse primer 361R 5’-CYIACTGCTGCCTCCCGTAG-3’ (Custom order–IDT)*
  *Note: These universal primers were first described in the article by Sundquist et al. (2007). Additionally, both forward and reverse primers need to include 5’ and 3’ linker sequences, heterogeneity spacers and the desired index identifier sequences that will allow dual indexing for later sample identification. These modifications are described in detail in the study of Fadrosh et al. (2014).
10. UltraPure agarose (Thermo Fisher Scientific, catalog number: 16500500 )
11. 50x TAE buffer (Bio-Rad Laboratories, catalog number: 1610743 )
12. GelPilot DNA Loading dye, 5x (QIAGEN, catalog number: 239901 )
13. UltraPure ethidium bromide (Thermo Fisher Scientific, catalog number: 15585011 )
14. 100 bp DNA ladder (New England BioLabs, catalog number: N3231S )
15. RNase AWAY decontamination reagent (Thermo Fisher Scientific, catalog number: 10328011 )
PCR Product Clean-up, quantification and sequencing
1. DNA low-bind tube 1.5 ml (Eppendorf, catalog number: 0030108051 )
2. Qubit assay tubes (Thermo Fisher Scientific, catalog number: Q32856 )
3. Agencourt AMPure XP (Beckman Coulter, catalog number: A63880 )
4. Ethanol for molecular biology (Sigma-Aldrich, catalog number: E7023 )
5. Buffer EB (QIAGEN, catalog number: 19086 )
6. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog number: Q32851 )
7. Experion DNA 1K Kit (Bio-Rad Laboratories, catalog number: 7007107 )
8. PhiX Control V3 (Illumina, catalog number: FC-110-3001 )
9. MiSeq Reagent Kit v3 (Illumina, catalog number: MS-102-3003 )
10. RNase AWAY decontamination reagent (Thermo Fisher Scientific, catalog number: 10328011 )

Equipment

Sterile scissors (VWR, catalog number: 82027-582 )
Sterile Fine scissors (Fine Science Tools, catalog number: 14058-11 )
Sterile medium tipped forceps (Fisher Scientific, catalog number: 12-000-157 )
Pipettes
UV crosslinker (UV Stratalinker 1800) (Stratagene, model: Model 1800 )
Microcentrifuge (Eppendorf, model: 5424 R )
Heated shaking block for Eppendorf tubes (Eppendorf, model: Thermomixer 5436 )
Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
Spectrophotometer (Thermo Fisher Scientific, model: NanoDrop^TM 1000 , catalog number: ND-1000)
Thermal Cycler (Bio-Rad Laboratories, model: DNA Engine )
Electrophoresis Chamber (Bio-Rad Laboratories, model: Wide Mini-Sub Cell GT Cell )
Gel Imager (Syngene, model: InGenius3 )
DynaMag-2 Magnet (Thermo Fisher Scientific, model: DynaMag^TM-2 Magnet )
Automated electrophoresis station (Bio-Rad Laboratories, model: Experion^TM Automated Electrophoresis System )
Experion priming station (Bio-Rad Laboratories, catalog number: 7007030 )
Experion vortex station II (Bio-Rad Laboratories, catalog number: 7007043 )
Qubit 3.0 Fluorometer (Thermo Fisher Scientific, model: Qubit 3.0 )

Software

Software Mothur (Schloss et al., 2009)
Software R (R Core Team, 2017)
Software RStudio (RStudio Team, 2015)

Procedure

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中文翻译

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如何引用

Abusleme, L., Hong, B., Hoare, A., Konkel, J. E., Diaz, P. I. and Moutsopoulos, N. M. (2017). Oral Microbiome Characterization in Murine Models. Bio-protocol 7(24): e2655. DOI: 10.21769/BioProtoc.2655.