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Updated: Jul 14, 2026
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
[Construction of eukaryotic expression vectors containing different haplotype cDNA of human CIITA gene]
Xiu-juan Bai1, Xu-qing Zhang, Xiao-jun Hong
1PLA Infectious Diseases Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Objective:
To construct eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene.
Method:
cDNA fragments of three different CIITA haplotypes were obtained by inducing one or two single nucleotide mutations of wild type recombinant plasmid EBS-NPL-CIITA cDNA, which correspond to two non-homonymy single nucleotide polymorphism (SNP) sites in the coding region of human CIITA gene, using overlap extension PCR site-directed mutagenesis technology. The above-mentioned three haplotype cDNAs were respectively cloned to EBS-NPL-CIITA linearized vectors. Positive clones were identified by colonial PCR and restriction endonuclease digestion and were sent to be sequenced. Then eukaryotic expression vectors containing four different haplotypes and an empty vector EBS-NPL were transfected into HepG2 cells respectively. HLA-DR was detected by indirect cell immunofluorescence technique.
Results:
The cDNA fragments of three different human CIITA haplotypes were successfully constructed, and the eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were obtained. No expression of HLA-DR was observed in the original HepG2 cells and empty vector transfected HepG2 cells and the expression of HLA-DR emerged in the HepG2 cells transfected with four eukaryotic expression vectors.
Conclusion:
The eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were successfully constructed, and they are essential for our further study of the functional differences of them.
