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A method for rapid demineralization of teeth and bones.

Cho A, Suzuki S, Hatakeyama J, Haruyama N, Kulkarni AB - Open Dent J (2010)

Bottom Line: The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate.This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest.However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase.

View Article: PubMed Central - PubMed

Affiliation: Gene Targeting Facility, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

ABSTRACT
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

No MeSH data available.


Related in: MedlinePlus

Difference in the time required for accelerated demineralization of the skulls from the older mice.Soft x-ray images showing the time required for complete demineralization of the skulls of mice of different ages subjected to the demineralization process at 42°C in 0.1M EDTA. Note that for complete demineralization, the skulls of both 2-week-old and 1-month-old mice required 3 to 4 days, while the skulls of 3-month-old and 1-year-old mice required 5 to 6 days. 0.1M EDTA was used as a demineralizing agent for all of the temperatures tested.
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Figure 6: Difference in the time required for accelerated demineralization of the skulls from the older mice.Soft x-ray images showing the time required for complete demineralization of the skulls of mice of different ages subjected to the demineralization process at 42°C in 0.1M EDTA. Note that for complete demineralization, the skulls of both 2-week-old and 1-month-old mice required 3 to 4 days, while the skulls of 3-month-old and 1-year-old mice required 5 to 6 days. 0.1M EDTA was used as a demineralizing agent for all of the temperatures tested.

Mentions: Longer time required for demineralization of mouse skulls from older mice: Having demonstrated that we can achieve a rapid demineralization at 42°C with 0.1M EDTA, we then investigated the effects of aging on the time required for complete demineralization of the skulls. The time required for a complete demineralization of the skulls of 2-week-old and 1-month-old mice was 3 to 4 days, whereas for 3-month-old and 1-year-old mouse skulls it was 5 to 6 days (Fig. 6).


A method for rapid demineralization of teeth and bones.

Cho A, Suzuki S, Hatakeyama J, Haruyama N, Kulkarni AB - Open Dent J (2010)

Difference in the time required for accelerated demineralization of the skulls from the older mice.Soft x-ray images showing the time required for complete demineralization of the skulls of mice of different ages subjected to the demineralization process at 42°C in 0.1M EDTA. Note that for complete demineralization, the skulls of both 2-week-old and 1-month-old mice required 3 to 4 days, while the skulls of 3-month-old and 1-year-old mice required 5 to 6 days. 0.1M EDTA was used as a demineralizing agent for all of the temperatures tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040998&req=5

Figure 6: Difference in the time required for accelerated demineralization of the skulls from the older mice.Soft x-ray images showing the time required for complete demineralization of the skulls of mice of different ages subjected to the demineralization process at 42°C in 0.1M EDTA. Note that for complete demineralization, the skulls of both 2-week-old and 1-month-old mice required 3 to 4 days, while the skulls of 3-month-old and 1-year-old mice required 5 to 6 days. 0.1M EDTA was used as a demineralizing agent for all of the temperatures tested.
Mentions: Longer time required for demineralization of mouse skulls from older mice: Having demonstrated that we can achieve a rapid demineralization at 42°C with 0.1M EDTA, we then investigated the effects of aging on the time required for complete demineralization of the skulls. The time required for a complete demineralization of the skulls of 2-week-old and 1-month-old mice was 3 to 4 days, whereas for 3-month-old and 1-year-old mouse skulls it was 5 to 6 days (Fig. 6).

Bottom Line: The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate.This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest.However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase.

View Article: PubMed Central - PubMed

Affiliation: Gene Targeting Facility, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

ABSTRACT
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

No MeSH data available.


Related in: MedlinePlus