Application of Novel Laser Capture Microdissection and RT-PCR to a Functional Analysis of Osteopontin in Mouse Growth Plate Cartilage

Abstract

The long bones of vertebrates, such as the tibiae and femurs of humans, extend in length by means of genotypic and phenotypic changes orchestrated by the chondrocytes comprising growth plate cartilage. Among the constituents synthesized by these cartilage cells, osteopontin (OPN), a phosphorylated glycoprotein, is thought to play a critical role in events leading to normal growth plate function and ultimate mineralization of the deeper zones of this cartilage region. The precise role of OPN, however, is uncertain with regard to mineralization, and present evidence supports the alternative possibilities that the protein may be either facilitative or inhibitory to mineral deposition. in order to investigate OPN function in a model growth plate, cartilage from normal 1-11 day old postnatal mice was examined by the novel techique of laser capture microdissection (LCM) followed by RT-PCR to obtain a measure of OPN gene expression by chondrocytes of known age and specific location in the plate. LCM permits identification of individual or clusters of cells within a tissue section and subsequent unique isolation (“capture”) of such cells for a variety of molecular analyses.In this study, mouse tibiae were dissected, placed in RNAlater (Ambion, Austin, TX) to preserve message, and stored at −20°C. Sections (5 μm thick) of fresh frozen developing epiphyseal growth plates were obtained in a cryostat maintained at −20°C, fixed briefly in 70% ethanol, and stained with eosin. Sections were examined in a Pixcell LCM system (Arcturus Engineering, Mountain View, CA) where chondrocytes were attached to the surface of polymer film substrates and lifted free of sections. in separate experiments, ∼200-1200 cells were captured and analyzed. Substrates were transferred to Eppendorf tubes containing RNA extraction buffer. RNA was extracted from cells by microisolation (Stratagene, La Jolla, CA), DNAse-treated, reverse-transcribed, and then subjected to PCR (40 cycles) with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). Ethidium bromide agarose gels revealed OPN mRNA from groups of chondrocytes isolated from whole cartilage and from resting, proliferating, and hypertrophic growth plate zones from the mouse tibiae. Brain cells captured by LCM from the same mouse sections served as positive controls and reactions containing no reverse transcriptase were negative controls. 18S rRNA was used as a marker for semiquantitation and standardization of expressed message from captured cells.