Biomedical subjects

A Braut

Publications and source records attributed to A Braut.

3 recordsLinked to original sources

PubMed2004

Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene.

Our previous studies have demonstrated that promoter-green fluorescent protein (GFP) transgenes can be used to identify and isolate populations of cells at the preosteoblastic stage (pOBCol3.6GFP) and at the mature osteoblastic stage (pOBCol2.3GFP) in living primary bone cell cultures. This strategy forms the basis for appreciating the cellular heterogeneity of lineage and relating gene function to cell differentiation. A weakness of this approach was the lack of a selective marker for late osteoblasts and mature osteocytes in the mineralized matrix. In this study, we have examined the expression of DMP-1 mRNA in murine marrow stromal and calvarial osteoblast cultures, and in bone, and calvaria in vivo. Furthermore, we have generated transgenic mice utilizing a mouse DMP1 cis-regulatory system to drive GFP as a marker for living osteocytes. Transgene expression was directed to mineralized tissues and showed a high correlation with the expression of the endogenous gene. Osteocyte-restricted expression of GFP was observed in histological sections of femur and calvaria and in primary cell cultures. Generation of this transgenic model will facilitate studies of gene expression and biological functions in these terminally differentiated bone cells.

Animals↗

PubMed2002

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Animals↗

PubMed2002

Col1a1-GFP transgene expression in developing incisors.

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of alpha 1(I) collagen in various tissues including odontoblasts.

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