Generation and expression analysis of BAC humanized mice carrying HLA‐DP401 haplotype

Abstract

Background 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. Methods 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. Results 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. Conclusions We generated several BAC transgenic mice, and analyzed the expression of HLA‐DPA1/DPB1 in those mice. A model of S aureus‐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.

Development of a humanized HLA‐A30 transgenic mouse model

“…As expected, approximately 50% transgenic and 50% non‐transgenic offspring was produced in our BAC founder animals. Five to 10 copies were found in these humanized transgenic mice (Figure 1B,C), as in most published reports 28,29,35 . Expression of exogenous gene was measured by reverse‐transcription polymerase chain reaction (RT‐PCR) with the specifically designed primer (Figure 1D).…”

“…We estimated BAC copy number by quantitative PCR in all transgenic lines, as previously described. 28,29,35 As expected, approximately 50% transgenic and 50% non-transgenic offspring was produced in our BAC founder animals. Five to 10 copies were found in these humanized transgenic mice (Figure 1B,C), as in most published reports.…”

“…Before performing real‐time PCR, DNA samples were quantified and diluted to 10 ng/μl. A standard curve of quantitative PCR data was produced as previously reported 28,29 . No‐template controls were included in each experiment, and all PCR reactions were carried out in triplicate.…”

“…Tail DNA was generated from mice tail biopsies conventionally with tissue DNA extraction kit (Trans, Beijing), as previously reported. 28 Before performing real‐time PCR, DNA samples were quantified and diluted to 10 ng/μl. A standard curve of quantitative PCR data was produced as previously reported.…”

“…A standard curve of quantitative PCR data was produced as previously reported. 28 , 29 No‐template controls were included in each experiment, and all PCR reactions were carried out in triplicate. Copy number was calculated using the following equation: estimated copy number = 2e ([ΔCt − y intercept]/slope) .…”