Shaozhe Yang1,2,3, Haijun Shi1,2,3, Xinran Chu1,2,3, Xiaoling Zhou1,2,3, Pingnan Sun4,5,6. 1. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou, 515041, People's Republic of China. 2. The Center for Reproductive Medicine, Shantou University Medical College, Shantou, 515041, People's Republic of China. 3. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, 515041, People's Republic of China. 4. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou, 515041, People's Republic of China. Pingnan_sun@yahoo.com. 5. The Center for Reproductive Medicine, Shantou University Medical College, Shantou, 515041, People's Republic of China. Pingnan_sun@yahoo.com. 6. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, 515041, People's Republic of China. Pingnan_sun@yahoo.com.
Abstract
OBJECTIVES: To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. RESULTS: The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. CONCLUSION: We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.
OBJECTIVES: To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. RESULTS: The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. CONCLUSION: We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.
Authors: Andrew M Intlekofer; Alan H Shih; Bo Wang; Abbas Nazir; Ariën S Rustenburg; Steven K Albanese; Minal Patel; Christopher Famulare; Fabian M Correa; Naofumi Takemoto; Vidushi Durani; Hui Liu; Justin Taylor; Noushin Farnoud; Elli Papaemmanuil; Justin R Cross; Martin S Tallman; Maria E Arcila; Mikhail Roshal; Gregory A Petsko; Bin Wu; Sung Choe; Zenon D Konteatis; Scott A Biller; John D Chodera; Craig B Thompson; Ross L Levine; Eytan M Stein Journal: Nature Date: 2018-06-27 Impact factor: 69.504