Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts.
OBJECTIVE
As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3).


METHODS
The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity.


RESULTS
In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules.


CONCLUSIONS
The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.
Marijanović
Inga
I
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA. ingamarijanovic@hotmail.com
Jiang
Xi
X
Kronenberg
Mark S
MS
Stover
Mary Louise
ML
Erceg
Ivana
I
Lichtler
Alexander C
AC
Rowe
David W
DW
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D019076:Transgenes / Q000235:genetics
D005819:Genetic Markers; D015500:Chloramphenicol O-Acetyltransferase