Title

Characterization of skeletal phenotype of a bone specific Dlx3 knock out mice

Date of Completion

January 2008

Keywords

Biology, Genetics|Biology, Cell

Degree

Ph.D.

Abstract

This project aimed to understand role of Dlx3 in bone formation and metabolism. We used Cre-LoxP principle to generate bone specific Dlx3 knock out mice. Mice carrying a 3.6 kb type I collagen fragment upstream to Cre recombinase transgene were used to drive Cre expression in bone. Use of this promoter prevented Cre mediated genomic rearrangement of floxed Dlx3 (FDlx3) allele (Dlx3 transgene with 2 LoxP sites flanking the gene) and embryonic lethality during early embryonic development. The bone specific Dlx3 knock out mice were homozygous for FDlx3 and carried one copy of Cre transgene (F/F, Cre+). A skeletal phenotype was not detectable by whole mount skeletal staining of knock out and age and sex matched control mice. However, quantitative measurement of long bone, vertebrae and calvaria using microCT detected significant increase in bone mass in the conditional knock out mice compared to the control group. Von Kossa staining of undecalcified bone sections supported this finding. The conditional knock out mice had lower number of osteoblasts that was statistically significant in male knock out mice. Analysis of serum markers Trap5b and CTX were done to see change in osteoclast differentiation and function respectively. No difference was observed in these markers, suggesting osteoclast differentiation and function were not affected in these mice. Bone formation rate and mineral apposition rate were not significantly different either. Gene expression was studied using RNA from cultured cells by quantitative PCR method and showed changes in the expression pattern of other Dlx genes and multiple bone specific genes including expression of osteocalcin (OCN), bone sialoprotein (BSP), Collagen I (Col1a1) and alkaline phosphatese. We did not find an osteoblast differentiation defect when studied in the cell culture. We believe that Dlx3 is required for bone metabolism but dispensable for osteoblast differentiation. We also believe that lack of Dlx3 and loss of its transcriptional control over target genes result in a higher bone mass in the conditional knock out mice. ^