建立趨化激素CXCL14基因轉殖小鼠

Abstract

乳腺組織會隨著每一次懷孕分娩過程而經歷細胞的快速增生、分化、生長休止及細胞凋亡的過程。雌性動物懷孕及泌乳時，乳腺上皮細胞受生乳素刺激合成乳汁蛋白。但當胎兒停止吮乳，乳汁的滯留會引發退乳，乳腺經歷大規模的細胞凋亡、組織重建，以恢復至懷孕前的狀態，有許多重要基因參與其中調控。過去本研究室利用RNA扣除法篩選小鼠退乳第4天與泌乳第15天的乳腺組織，發現數個有表現差異的基因，CXCL14為在退乳期大量表現基因的其中之一，此結果與另一英國團隊以微陣列方式所得相同，他們發現CXCL14會在第三天達到表現高峰。 CXCL14除了有趨化免疫細胞的功能，也發現它會影響腫瘤生長，並且可能參與在胰島素的訊息傳遞路徑中。因此推測CXCL14可能會參與在第二階段的退乳中，透過吸引免疫細胞前來清除死亡細胞、或是促進乳腺上皮細胞增生。本實驗的目的是建立提早在泌乳期大量表現CXCL14的基因轉殖小鼠，以研究CXCL14於正常乳腺之功能。 首先為了先建立受生乳激素誘導表現CXCL14的基因組，所以將乳汁中主要成分的β酪蛋白的啟動子序列與CXCL14基因融合，構築於哺乳類動物細胞表現載體上，將其轉染至乳腺上皮細胞中，以含有泌乳素、氫皮質酮的培養液誘導β酪蛋白啟動子活化，最後以細胞免疫螢光染色法及西方吸漬法分析，驗證外源CXCL14可成功於乳腺上皮細胞表現。從質體上分離轉殖基因片段，並委託國家實驗動物中心產製基因轉殖小鼠。從國家實驗動物中心獲得10隻基因轉殖小鼠後，皆分別獨立與野生型小鼠配種，其子代利用PCR方法進行基因型鑑定。最後共有6隻可順利將轉殖基因片段傳至子代；有2隻無法將轉殖基因傳遞到子代、2隻則是不易懷孕或有食仔癖導致尚無得到帶有轉殖基因之子代。 綜合上述結果，我們已成功建立受生乳激素誘導表現CXCL14的基因組，經證實可在哺乳類乳腺上皮細胞中表現後，也順利得到基因轉殖小鼠。但轉殖基因的重複數、嵌入體染色體點位數，以及轉殖基因是否對乳腺發育有影響皆須再檢測。一旦成功建立此基因轉殖小鼠，未來可提供作為研究CXCL14對乳腺發育影響之利器。

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.