| Summary: | 碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 103 === Mammary gland will undergo cell proliferation, differentiation, growth arrest and cell apoptosis during each pregnancy. Fully differentiated mammary epithelial cells would be stimulated by prolactin and hydrocortisone and synthesize milk proteins. If the pups do not suckle any more, milk accumulation in the ducts will trigger cell apoptosis occurrence. Tissue remodeling follows the large-scale cell death and finally the mammary gland will return to the state before pregnancy. There are a lot of genes involved the regulation of mammary gland development. By using PCR-select cDNA subtraction to select genes differentially expressed in mouse mammary gland in lactating day 15 and involution day 4, a chemokine celled CXCL14 was identified.
CXCL14, a member of CXC chemokine, has been described not only to play a role in trafficking immunocytes, just like the other chemokines, but also be a tumor suppressor or enhancer, and involved in glucose metabolism. Based on these finding, CXCL14 might participate the event during involution, including the immune cell trafficking, mammary epithelial cell survival and adipocyte re-proliferation. Therefore, the aim of this study is to generate transgenic mice containing a lactogenic hormone-inducible cassette to overexpressing CXCL14 during lactation, which forced its expression earlier.
In order to construct a lactogenic hormone-inducible cassette, the promoter of β-casein, one of the major components of milk proteins, was fused with CXCL14 gene. The plasmid was transfected into mammary gland epithelial cells, and cultured with induction medium containing prolactin and hydrocortisone to examine the feasibility of the construction. The expression of CXCL14 was confirmed by western blotting. In the result of immunostaining, CXCL14 protein distributed in cytoplasm, that was the characteristic of secretory proteins. Finally, the cassette was as isolated to produce transgenic mice, which was executed by National Laboratory Animal Center (NLAC). 10 transgenic founders were generated from NLAC. Each founder bred separately, and the offspring was screened with PCR genotyping method. There are 5 founders transmitting the transgene to the offspring, and the other 5 neither raised up the offspring nor been pregnant smoothly.
In summary, we have constructed a lactogenic hormone-inducible CXCL14 gene cassette, proven its expression in mammary gland epithelial cell in vitro and bred the transgenic mice. The integration number and copy number of the transgene and its effect are still need to be examined. Once upon the transgenic mice are generated successfully, it will be a useful material to study the role of CXCL14 in mammary gland development.
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