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Updated: Jun 6, 2026
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
[Construction of human nm23-H1 mutant and EGFP fusion genes using site-directed mutagenesis]
Daxing Zhu1, Qinghua Zhou, Yanping Wang
1Key Laboratory of Lung Cancer Molecular Biology of Sichuan Province, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R.China.
Background:
Previous researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene.
Methods:
Site-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange™ Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR.
Results:
Five nm23-H1 mutant and EGFP fusion genes, nm23-H1(S44A)-EGFP, nm23-H1(P96S)-EGFP, nm23-H1 (H118F)-EGFP, nm23-H1(S120G)-EGFP and nm23-H1(P96S-S120G)-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design.
Conclusions:
Five nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange™ site-directed mutagenesis is a simple, rapid and efficient method.
