Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Summary

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

Abstract

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX₃CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

Introduction

Humans and bacteria have coexisted for a long time. They have established a codependent relationship with mutual beneficial effects that influences host immune responses in quantitative and qualitative ways¹. Recent studies suggest an association between the gut microbiota composition and the pathogenesis of autoimmune diseases that include multiple sclerosis², rheumatoid arthritis³, type 2 diabetes⁴, Inflammatory bowel disease⁵, and systemic lupus erythematosus (SLE)⁶. However, whether the gut microbiota is the main cause or ....

Protocol

The Cx3cr1^gfp/gfp locus of B6.129P2(Cg)-Cx3cr1^tm1Litt/J mice was backcrossed to MRL/MpJ-Fas^lpr/J (MRL/lpr) for 10 generations to generate MRL/lpr-CX₃CR1^gfp/gfp mice. Genome screening using single nucleotide polymorphism (SNP) panels confirmed that the genetic background of newly generated mice was more than 97% identical to that of MRL/lpr. After that, mice were bred and maintained in a specific pathogen-free environment following the specific requi.......

Discussion

Balanced gut microbiota can protect the human body from diseases. Once this balance is disrupted by external or internal triggers, consequences can be devastating. This method presents a way to analyze the dynamics of gut microbiota in murine models. The method is suitable not only for comparisons among groups but also for tracking the gut microbiota over time to better identify time-dependent factors disrupting the gut microbiota.

All the mice in the experiment must be handled in the same env.......

Materials

Name	Company	Catalog Number	Comments
0.1 mm glass beads	BioSpec Products	11079101
2 mL screw cap tubes	Thermo Fisher Scientific	3488
20% SDS	FisherScientific	BP1311-1	SDS 20%
96% Ethanol, Molecular Biology Grade	Thermo Fisher Scientific	T032021000CS
Ammonium Acetate (5 M)	Thermo Fisher Scientific	AM9071	NH4AC 5M
B6.129P2(Cg)-Cx3cr1tm1Litt/J	Jackson Laboratory	005582
Bullet Blender storm 24	Next Advance	4116-BBY24M	Homogenizer
Chloroform	FisherScientific	C298-500
DEPC-Treated Water	Thermo Fisher Scientific	AM9916
Ethylenediamine Tetraacetic Acid	FisherScientific	BP118-500	EDTA
Foil plate seal	FisherScientific	NC0302491
Kimwipes-Kimtech 34256	FisherScientific	06-666C
MRL/MpJ-Faslpr/J (MRL/lpr) mice	Jackson Laboratory	000485
Nanodrop 2000 spectrophotomer	Thermo Fisher Scientific	ND2000CLAPTOP
Phenol: chloroform: isoamylalchohol (25:24:1)	FisherScientific	BP1752I-400	PCI
Scale with 4 decimals	Mettler Toledo	MS205DU
Skirted 96-well plates	Thermo Fisher Scientific	AB-0800
Sodium chloride	FisherScientific	15528154	NaCl
Tris Hydrochloride	FisherScientific	BP1757-100
Vortex	Scientific Industries	SI-0236