Microarray Profiling the Murine Osteoprogenitor Lineage

University of Connecticut Health Center, Farmington, CT
University of Connecticut, Storrs CT
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Because bone is a self-renewing tissue, the effectiveness of its progenitor lineage in generating a critical number of mature osteoblasts is a major determinant of adult bone mass. Thus assessing the performance of the lineage and the molecular genetic events that are crucial to lineage progression is an essential element in understanding murine models of bone mass. This workshop will present an experimental strategy that can be utilized to meet these two requirements for explaining disease models of bone mass. It will also introduce a model that utilizes these tools for odontoblast differentiation.

WORKSHOP OBJECTIVES:
1. Present the concepts and fluorescent-based reagents used to identify cells at increasing levels of osteoblast/odontoblast differentiation in primary culture and intact murine tissue.
2. Utilize the tools and strategies for a microarray analysis of the osteoblast lineage using a bone/tooth-optimized cDNA library.

  

WORKSHOP OUTLINE:   Back to Top

A. Prior to the conference

To make this learning experience relevant to your experimental model of bone, the participant is encouraged to(but is not required):
  1. Submit any paraformaldehyde fixed tissues harboring a GFP reporter that you would like to view after cryosectioning.
  2. Submit paraformaldehyde-fixed dissected intact and nondecalcified femurs from a fluorescent-labeled (calcein and xylenol orange) mice for histological analysis.
  3. Propose a microarray experiment that you could supply RNA by March 1 (see web application form). We will pick two proposals from the submitted proposals with the expectation that the RNA from the experiments will be delivered to us by April 1. The results of two submitted samples will be used for teaching purposes.
  4. Indicate any special preferences that you would like emphasized from the list of optional topics. Prior to arrival, a plan will be developed for the two individuals per laboratory to rotate to a different instructor to cover each selected topic. The topic will be repeated for each of the 10 laboratory participants so that personalized attention will be paid to each participant. One day will be focused on the cell biology of bone lineage and the other on the microarray analysis of the lineage.

B. Cell biology day (Univ. of CT Health Center at Farmington)

1. Analysis of lineage in histological sections of murine bone/teeth

1. Jiang X,et.al. Histological analysis of GFP expression in murine bone. J Histochem Cytochem. 2005 May;53(5):593-602.
  2. Bilic-Curcic I et.al. Visualizing levels of osteoblast differentiation by a two-color promoter-GFP strategy: Type I collagen-GFPcyan and osteocalcin-GFPtpz. Genesis. 2005 Oct;43(2):87-98.
  3. Kalajzic I, et.al. Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene. Bone. 2004 Jul;35(1):74-82.


 
•
 Embedding and full length sectioning of frozen decalcified and non-decalcified bone using the CryoJane tape transfer method.
   
  • Fluorescent –based stains for in vivo mineralization, alkaline phosphatase, TRAP, TUNEL, cell nuclei, and total mineralized bone will be demonstrated on the bone you provide.
   
  • Multiplex-imaging techniques for visualizing GFP (5 different colors) in association with the fluorescent-based stains.
   
  • Image processing to generate full length scans of bone and to produce merged overlapping images.
   


2. Analysis of lineage in primary bone cell/dental pulp culture (calvarial, marrow stromal, dental pulp and bone chips)

  1. Wang YH. et.al. Comparison of the action of transient and continuous PTH on primary osteoblast cultures expressing differentiation stage-specific GFP.J Bone Miner Res. 2005 Jan;20(1):5-14.
  2. Kalajzic, I. et.al. Stage specific inhibition of osteoblast lineage differentiation by FGF2 and noggin. J Cell Biochem. 2003 Apr 15;88(6):1168-76.
  3. Kalajzic I. et.al. Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage. J Bone Miner Res. 2002 Jan;17(1):15-25.

 
•
 Obtain the temporal GFP expression and nodule mineralization pattern of maturing bone or dental pulp cell culture.
   
  • FACS analysis of cultured primary bone cells for co-expression of GFP with fluorescent markers of apoptosis and cell division.
   
  • FAC sorting to isolate a subpopulation of cells from primary osteoblast culture.

3. Additional topics can be specifically selected

  1. Wang L. et.al. Heterogeneity of engrafted bone-lining cells after systemic and local transplantation. Blood. 2005 Nov 15;106(10):3650-7.
  2. Bilic-Curcic I. et.al. Origins of endothelial and osteogenic cells in the subcutaneous collagen gel implant. Bone. 2005 Nov;37(5):678-87.
  3. Stover ML. et.al. Bone-directed expression of Col1a1 promoter-driven self-inactivating retroviral vector in bone marrow cells and transgenic mice. Mol Ther. 2001 Apr;3(4):543-50.

 
•
 Quantitation of fluorescent signals within the images from cell culture or bone histology.
  • Use of GFP marked donor and recipient mice to interpret the stem cell properties of a transplanted progenitor population.
   
  • GFP transgenic mice that mark the endothelial, pericyte, osteoclast, chondrocyte, myoblast and adipocyte lineages.
  • Retroviral and lentiviral transduction of primary cells with cDNA expression or RNAi constructs
  • Construct design for traditional and BAC transgenic mice
  • Mouse colony management – cage side database data recording, genotyping by cage side fluorescence goggles, qPCR method for transgene gene dosage.
   




 

C. Microarray day (Computer Science Laboratory, Univ. of CT at Storrs)

  Kalajzic I. et.al. Expression profile of osteoblast lineage at defined stages of differentiation. J Biol Chem. 2005 Jul 1;280(26):24618-26.

1. Technical aspects of a cDNA microarray study

 
•
 Current and emerging platforms
cDNA platform: Selection of genes, amplification and spotting
 
•
 Sample quality, labeling and hybridization
 
•
 Obtaining intensity values from the hybridization


2. Data processing – preparing the intensity value for analysis

 
•
 Techniques for background correction, normalization and imputation
 
•
 Array utility: a program for automated data processing for Bioconductor

3. Statistical evaluation of the microarray study using your data.

  Woo Y. et.al. Experimental design for three-color and four-color gene expression microarrays. Bioinformatics. 2005 Jun 1;21 Suppl 1:i459-i467.

 
•
 Reference vs loop designed microarray study
Statistical analysis of microarray (SAM)
 
•
 Semiparametric hierarchical Bayesian (SPHB)
 
•
 LIMMA and MAANOVA

4. Annotation and viewing the results of your microarray study

 
•
 Regulated gene lists
 
•
 Display Utility: A Shared database for group annotation
  • Magellan: Mapping data to genetic pathways
 
•
 Mapping data to pathways – GenMapp, http://www.genmapp.org/


5. Computational tools for gene clustering

  Garg S. et.al. An adaptive strategy for single- and multi-cluster gene assignment. Biotechnol Prog. 2003 Jul-Aug;19(4):1142-8.

 
•
 Adaptive centroid algorithm: application to expression data and GO function.

6. Publishing your microarray study

 
•
 MIAME
 
•
 MAGE-ML
 
•
 Gene Expression Omnibus (GEO), http://www.ncbi.nlm.nih.gov/geo/

WORKSHOP TIMELINE    Back to Top

A weekend in early May, 2006 has been selected for availability of the computer laboratories (Storrs campus) and sufficient time for processing of samples for histological examination.

  February 1 Registration Opens
  March 15 Submit samples for histology
  April 1 Submit samples for microarray
  April 15 Registration payment received
  May 1 Topics selected and teaching plans finalized
  May 12 Late afternoon arrival; orientation and social gathering
  May 13 Day 1 of instruction. Free Saturday evening.
  May 14 Day 2 of instruction.

REGISTRATION AND ANTICIPATED EXPENSES   Register Now  Back to Top

The conference is designed to accommodate two individuals from a single laboratory. You will rotate together to the individual instruction stations to reinforce and explore how the presented material relates to your research program. The cost of the workshop is $500 per individual or $900 per laboratory pair. This expense will cover:

  • Hotel room accommodations for each participant on May 12 and May 13 (West Hartford Inn, West Hartford, CT).
Friday evening meal on May 12.
 
  • Breakfast, lunch and group dinner on May 13.
  • Breakfast and lunch on May 14.
  • Transportation from the hotel to Farmington (cell biology) and to Storrs (microarray) on both days of the workshop.
 
  • We will try to provide transportation for the return leg of your trip.
  • Cost for histology and microarray study.

You will be responsible for:

  • Transportation to and from Hartford including your connection from the airport to the hotel in West Hartford.

Registration will be through this web site. Questions can be directed to Ms. Michelle Proper at 860-679-1191 or by email to molcore@nso1.uchc.edu.

WORKSHOP SPONSORS:    Back to Top

Support to Dr. David Rowe and Dr. Dong-Guk Shin for this workshop includes:

NIDDK R13 DK070516-01, Workshop for microarray profiling the osteoblast lineage;
NIDDK U01 DK63478, The osteoprogenitor lineage within the Resource Development for the Gene Anatomy Project ;
NIAMS P30 46026, Microarray Facility within the Core Center for Musculoskeletal Research;
NIGMS P20 GM65764, Bone Cell Differentiation Project with the Integrated bioinformatics center of cellular biology.