Related Experiment Videos
Porcine MCP gene promoter directs high level expression of human DAF (CD55) in transgenic mice
H Murakami1, Y Takahagi, M Yoshitatsu
1Research and Development Center, Nippon Meat Packers, Inc., Tsukuba, Ibaraki, Japan.
Abstract:
Porcine membrane cofactor protein (pMCP), a complement regulatory protein, is widely expressed in various tissues. Particularly, it is highly expressed on vascular endothelium. The objective of this study was to investigate whether the pMCP gene promoter can induce efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55) in transgenic mice. Two fragments of the 5'-flanking region of pMCP gene (0.9 kb and 5.4 kb) connected with human DAF minigene (0.9/hDAF and 5.4/hDAF) were used to produce transgenic mice. The expression of hDAF in heart, liver, kidney, lung, pancreas, brain and testis of the transgenic mice was examined by immunohistochemical analysis. The vascular endothelia and the nerves in all organs examined were intensely stained. The staining pattern in these tissues was similar in all transgenic mice examined regardless of the length of the promoters. The surface expression levels of hDAF on peripheral red blood cells and splenocytes from a mouse carrying 5.4/hDAF hemizygously was twice the level of expression on corresponding human cells. The red blood cells and splenocytes from the transgenic mice exhibited resistance to lysis by human serum in a manner dependent upon expressed hDAF level. The hearts from the transgenic mice functioned for a significantly longer time than those from normal mice under perfusion with human serum in the Langendorff perfusion system. These results demonstrated that the pMCP gene promoter is a good candidate of the regulatory element in the transgene to produce transgenic animals for xenotransplantation.
Insights
The porcine membrane cofactor protein (pMCP) gene promoter efficiently drives expression of human decay-accelerating factor (DAF) in transgenic mice. This finding supports pMCP promoter use for xenotransplantation.
Area of Science:
- Immunology
- Genetics
- Transplantation Biology
Background:
- Porcine membrane cofactor protein (pMCP) is a complement regulatory protein highly expressed on vascular endothelium.
- Efficient expression of complement regulatory proteins is crucial for xenotransplantation success.
Purpose of the Study:
- To investigate the potential of the pMCP gene promoter to drive expression of human decay-accelerating factor (DAF; CD55) in transgenic mice.
- To assess the functional consequences of DAF expression in transgenic mice for xenotransplantation.
Main Methods:
- Generation of transgenic mice using pMCP promoter fragments (0.9 kb and 5.4 kb) linked to a human DAF minigene.
- Immunohistochemical analysis of hDAF expression in various organs of transgenic mice.
- Flow cytometry to quantify hDAF surface expression on red blood cells and splenocytes.
- Assessment of resistance to human serum lysis and ex vivo heart function in a Langendorff perfusion system.
Main Results:
- The pMCP promoter fragments successfully directed hDAF expression in vascular endothelium and nerves across multiple organs.
- Transgenic mice exhibited elevated hDAF surface expression on blood cells compared to human cells.
- Cells and organs from transgenic mice showed resistance to human serum-mediated lysis and improved function.
Conclusions:
- The pMCP gene promoter is an effective regulatory element for driving transgene expression in mice.
- This promoter is a promising candidate for developing transgenic animals for xenotransplantation, enhancing complement-mediated protection.