The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Summary

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

Abstract

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

Introduction

The master clock in mammals, which orchestrates 24 h oscillations for nearly all aspects of physiology and behavior, is located within the suprachiasmatic nucleus (SCN) of the hypothalamus^1,2. In addition to regulating biological processes on an organismal level, the master clock also synchronizes peripheral cellular clocks throughout the body^3,4,5. While the molecular clock machinery consists of at least three interlocking transcriptional-translational feedback loops, the core is comprised of the Period (

Protocol

During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the University of Hartford Animal Institutional Animal Care and Use Committee.

1. Entrainment of Animals

NOTE: Twenty week-old male B6129SF2/J mice are used in the study.

1. Entrain mice to a 12 h light (standard overhead white light)/12 h dark cycle for 2 weeks prior to the experiment.
   NOTE: Here zeitgeber time (ZT) 0 corresponds to lights on and ZT12 to lights off, while keeping all other environmental factors (i.e., food,....

Results

Mice were sacrificed at ZT13, splenocytes were isolated and challenged ex vivo with the PAMPs LPS, ODN1826, or HKLM. After 3 h, RNA was isolated, and qPCR was used to assess relative expression levels of the molecular clock genes Clock, Per2, Dbp, and Rev-erbα compared to unchallenged control cells. After PAMP challenge, Clock expression levels were not significantly different than expression in the control cells (.......

Discussion

Within this protocol, a microvolume spectrophotometer can be used to quantify and assess the purity of the RNA being used in determining gene expression. Nucleic acids absorb UV light at 260 nm, proteins typically absorb light at 280 nm, while other potential contaminants used during an RNA extraction procedure (e.g., phenol) are detectable at 230 nm. Therefore, by assessing the absorbance (A) ratio at 260/280 nm (RNA to protein) and 260/230 nm (RNA to non-protein contaminants) the quality of the RNA can be asse.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Frosted slides	Fisher	12-550-343
Cell strainers	Fisher	22363547
Lipopolysaccharide	InvivoGen	ltrl-eklps
ODN1826	InvivoGen	Tlrl-1826-1
HKLM	InvivoGen	Tlrl-hklm
RPMI 1640	Gibco	11875-093
PBS	Gibco	20012-043
RNeasy Mini Kit	Qiagen	74104 or 74106
RNase-Free DNase Set	Qiagen	79254
6-well cell culture plate	Denville	T1006
50 mL tubes	Corning	352070
15 mL tubes	Corning	352097
High Capacity cDNA Reverse Transcription Kit	ThermoFisher	4368814
TaqMan Gene Expression Assays b-actin	ThermoFisher	Mm00607939_s1
TaqMan Gene Expression Assays Per2	ThermoFisher	Mm00478113_m1
TaqMan Gene Expression Assays Rev-erba	ThermoFisher	Mm00520708_m1
TaqMan Gene Expression Assays Bmal1	ThermoFisher	Mm00500226_m1
TaqMan Gene Expression Assays Dbp	ThermoFisher	Mm00497539_m1
qPCR machine StepOnePlus	ThermoFisher
TaqMan Gene Expression Master Mix	ThermoFisher	4369016
MicroAmp Fast 96-well reaction plate (0.1 mL)	ThermoFisher	4346907
Statistical Analysis Software	Prism 7.0a