Generation of Erp60 Knockout Mice
Introduction:
1 Vitamin D is critical for embryonic skeletal formation and long bone growth. Without vitamin D, the hypertrophic cell zone will expand because the cartilage fails to calcify leading to rickets. Protein disulfide isomerase A3 (PDIA3), also known as ERp60, ERp57, Grp58, and 1,25-MARRS, is a multifunctional protein that has been associated with rapid membrane-initiated signaling by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in several cell types. Knockdown of PDIA3 in osteoblast-like cell line MC3T3 showed that reduction of PDIA3 expression led to remarkable decrease of PKC activation with 1,25 (OH) 2VitD3 treatment as well as a decrease in expression of several osteogenic genes compared to wild type cells, which indicated the important role of PDIA3 in vitamin D-regulated cartilage and bone development. However, the mechanism involved still remains unclear. The fact motivate us to generate the PDIA3 knockout mice which will serve as a good model to further explore the physiological function of PDIA3 and to understand the mechanism involved in PDIA3-mediated 1,25VitD3 signal pathway.
To date, the major problems we are facing during the generation an ERp60 Knockout mice is the lack of a liable genotyping method to distinguish the Knockout mice from the heterozygous mice. No knockout mice available from the previous breeding along with very lower percentage of wild type mice which did not match to the Mendel’s rule of Segregation. Therefore, the object of my project is firstly to establish a new and reliable method for genotyping, Secondly, to confirm the ablation of PDIA3 at protein level. Finally, the third objective is to explore the structural and functional abnormality in the PDIA3 knockout mice.
Methods:
DNA extraction and purification:
Mice tail clips were sliced into small pieces to increase the surface area and were placed in an Eppendorf tube with 300 uL Lysis Buffer and 30 ul protinease K for each tail clip. The Eppendorf tubes
1 Vitamin D is critical for embryonic skeletal formation and long bone growth. Without vitamin D, the hypertrophic cell zone will expand because the cartilage fails to calcify leading to rickets. Protein disulfide isomerase A3 (PDIA3), also known as ERp60, ERp57, Grp58, and 1,25-MARRS, is a multifunctional protein that has been associated with rapid membrane-initiated signaling by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in several cell types. Knockdown of PDIA3 in osteoblast-like cell line MC3T3 showed that reduction of PDIA3 expression led to remarkable decrease of PKC activation with 1,25 (OH) 2VitD3 treatment as well as a decrease in expression of several osteogenic genes compared to wild type cells, which indicated the important role of PDIA3 in vitamin D-regulated cartilage and bone development. However, the mechanism involved still remains unclear. The fact motivate us to generate the PDIA3 knockout mice which will serve as a good model to further explore the physiological function of PDIA3 and to understand the mechanism involved in PDIA3-mediated 1,25VitD3 signal pathway.
To date, the major problems we are facing during the generation an ERp60 Knockout mice is the lack of a liable genotyping method to distinguish the Knockout mice from the heterozygous mice. No knockout mice available from the previous breeding along with very lower percentage of wild type mice which did not match to the Mendel’s rule of Segregation. Therefore, the object of my project is firstly to establish a new and reliable method for genotyping, Secondly, to confirm the ablation of PDIA3 at protein level. Finally, the third objective is to explore the structural and functional abnormality in the PDIA3 knockout mice.
Methods:
DNA extraction and purification:
Mice tail clips were sliced into small pieces to increase the surface area and were placed in an Eppendorf tube with 300 uL Lysis Buffer and 30 ul protinease K for each tail clip. The Eppendorf tubes