人类白细胞抗原(HLA)-DP比其他HLAII类抗原的研究要少得多,也就是说,HLA-DR和HLA-DQ,等。然而,积累的数据表明DP限制性反应在癌症中的重要作用,过敏,和传染病。缺乏将这些基因表达为真正的顺式单倍型的动物模型阻碍了我们对HLA-DP单倍型在免疫中的作用的理解。
探索参与HLA-DPA1/DPB1基因转录调控的潜在顺式调控元件,我们在C57BL/6背景下使用基于细菌人工染色体(BAC)的转基因人源化小鼠进行表达分析,携带完整的HLA-DP401基因位点。我们进一步建立了HLA-DP401人源化转基因小鼠金黄色葡萄球菌肺炎小鼠模型,并对模型中HLA-DP401的表达模式和免疫应答进行了分析。
在这项研究中,我们筛选并鉴定了跨越整个HLA-DP基因位点的BAC克隆。通过脉冲场凝胶电泳分析来自该克隆的DNA的完整性,然后将其显微注射到受精的小鼠卵母细胞中以产生转基因建立动物。使用九组用于区域标记的PCR引物,每个引物之间的平均距离为15kb,以确认携带HLA-DPA1/DPB1基因的五个转基因品系中转基因的完整性。通过实时PCR分析确定转基因拷贝数。在mRNA和蛋白质水平分析HLA-DP401基因表达。尽管感染金黄色葡萄球菌纽曼并没有改变HLA-DP401-H2-Aβ1人源化小鼠的脾和胸腺中免疫细胞的百分比。在感染金黄色葡萄球菌的人源化小鼠的胸腺中观察到HLA-DP401的表达增加。
我们产生了几只BAC转基因小鼠,并分析了HLA-DPA1/DPB1在这些小鼠中的表达。进一步建立HLA-DP401-H2-Aβ1-/-人源化小鼠金黄色葡萄球菌肺炎模型,和金黄色葡萄球菌感染上调这些人源化小鼠胸腺中HLA-DP401的表达。这些发现证明了那些HLA-DPA1/DPB1转基因人源化小鼠用于开发感染性疾病和MHC相关免疫疾病的动物模型的潜力。
Human leukocyte antigen (HLA)-DP is much less studied than other HLA class II antigens, that is, HLA-DR and HLA-DQ, etc. However, the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity.
To explore the potential cis-acting control elements involved in the transcriptional regulation of the HLA-DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)-based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA-DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA-DP401 and immunological responses in the model.
In this study, we screened and identified a BAC clone spanning the entire HLA-DP gene locus. DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene. Transgene copy numbers were determined by real-time PCR analysis. HLA-DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aβ1 humanized mice. Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus.
We generated several BAC transgenic mice, and analyzed the expression of HLA-DPA1/DPB1 in those mice. A model of Saureus-induced pneumonia in the HLA-DP401-H2-Aβ1-/- humanized mice was further developed, and S aureus infection upregulated the HLA-DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.