Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Summary

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Abstract

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 ^fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Introduction

Skeletal bone is the main load-bearing organ of the human body and is under pressure from both the internal and external environment during walking and exercise¹. Throughout one’s life, bones continuously go through self-renewal, which is balanced by osteoblasts and osteoclasts. The process of osteoclasts clearing old bones and osteoblasts forming new bone maintains the homeostasis and mechanical function of the skeletal system². Disturbance in the balance may induce bone metabolic diseases, such as osteoporosis. Osteoporosis, which is caused by excess osteoclastic activity, is globally prevalent and causes substan....

Protocol

All methods relating to the animals described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University School of Medicine.

1. Breeding of osteoclast specific Stat3 deletion mice

NOTE: Stat3^fl/fl mice were obtained commercially. Ctsk-Cre mice were provided by S. Kato (University of Tokyo, Tokyo, Japan¹²). The mice were bred and maintained under specific pathogen-free (SPF) conditions in the institutional animal facility under standardized conditions.

1. Pair a sexually mature male mo....

Results

Using the present protocol, osteoclast specific Stat3 deletion mice were generated to study the influence of STAT3 deletion on osteoclast differentiation. Stat3^Ctsk mice and their wildtype (WT) littermates were bred and kept after genotyping. Bone marrow macrophages were isolated and cultured into osteoclasts, and STAT3 deletion in Stat3^Ctsk mice was demonstrated (Figure 1).

Femora reconstruction a.......

Discussion

Genetically engineered mouse models are commonly used for studying the mechanism and pharmaceutical treatment of human disease¹³. Ctsk-Cre mice have been widely used for functional studies of osteoclasts⁶. The present study described the protocols of the methods to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Histological analysis is the best intuitive method to detect bone metabolis.......

Materials

Name	Company	Catalog Number	Comments
4% Paraformaldehyde solution	Sangon biotech Co., Ltd.	E672002
Acetone	Shanghai Experimental Reagent Co., Ltd.	80000360
Alizarin	Sigma-Aldrich	A5533
Ammonia solution	Shanghai Experimental Reagent Co., Ltd.
Calcein	Sigma-Aldrich	C0875
Ctsk-Cre mice			a gift from S. Kato, University of Tokyo, Tokyo, Japan
DDSA	Electron Microscopy Sciences	13710
DeCa RapidlyDecalcifier	Pro-Cure	DX1100
DMP-30	Electron Microscopy Sciences	13600
EDTA	Shanghai Experimental Reagent Co., Ltd.	60-00-4
EMBED 812 RESIN	Electron Microscopy Sciences	14900
fluorescence microscope	Olympus	IX73
Hematoxylin solution	Beyotime Biotechanology	C0107
Micro-CT	Scanco Medical AG	μCT 80
NaHCO3	Shanghai Experimental Reagent Co., Ltd.	10018918
Neutral balsam	Sangon biotech Co., Ltd.	E675007
NMA	Electron Microscopy Sciences	19000
Paraffin	Sangon biotech Co., Ltd.	A601889
rotary microtome	Leica	RM2265
Stat3fl/fl mice	GemPharmatech Co., Ltd	D000527
TRAP staining kit	Sigma-Aldrich	387A
xylene	Shanghai Experimental Reagent Co., Ltd.	1330-20-7