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Published: 2015-02-12

Screening of HaCaT Clones for CCL20 Gene Knockout and Preliminary Exploration of Gene-Targeting Vector Transfection Approaches in this Cell Line

Yong Wang, Daizhi Peng, Lihua Wang, Zhengxue Dong, Bing He

(Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China (mainland))

Med Sci Monit Basic Res 2015; 21:21-28

DOI: 10.12659/MSMBR.893143


Background: Inhibition of CC chemokine ligand 20 (CCL20), which is expressed by human keratinocytes after proinflammatory cytokine stimulation, may reduce migration of recipient Langerhans cells into tissue-engineered allogeneic skin grafts and minimize immune rejection by the recipient. Here, we screened CCL20 gene knockout clones in the human immortalized skin keratinocyte line HaCaT and tested multiple transfection methods for optimal efficiency.
Material and Methods: The CCL20 gene was PCR amplified from HaCaT genomic DNA. Both the short arm (1,969 bp) and long arm (2,356 bp) of human CCL20 were cloned into ploxP-targeting vectors at either side of the neomycin resistance cassette, respectively. The resulting ploxP-hCCL20-targeting vector was linearized and electroporated into HaCaT. The positive HaCaT clones were screened under the pressure of both G418 and GANC, and identified by PCR and Southern blot. The ploxP-hCCL20-EGFP fluorescent expression vector was also constructed and transfected into 293FT and HaCaT cells by jetPEI liposome and nucleofection electroporation for evaluating the transfect efficiency under fluorescent microscope.
Results: The replacement targeting vector ploxP-hCCL20 (11.9 kb) for exon 2 of the human CCL20 gene was successfully constructed and transfected into HaCaT cells. The selected HaCaT clones did not show any evidence of CCL20 gene knockout by either PCR or Southern blot analysis. We also successfully constructed a fluorescent expression vector ploxP-hCCL20-EGFP (13.3 kb) to assess possible reasons for gene-targeting failure. Transfection efficiencies of ploxP-hCCL20-EGFP into 293FT and HaCaT cell li... read more

Keywords: Chemokine CCL20, Gene Targeting, Keratinocytes, Transfection




Published: 2015-02-09

Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry


Kuan Liu, Peng-cheng Liu, Run Liu, Xing Wu

Med Sci Monit Basic Res 2015; 21:15-20

DOI: 10.12659/MSMBR.893327

Published: 2015-02-03

Ex Vivo Lung Perfusion – State of the Art in Lung Donor Pool Expansion


Aron-Frederik Popov, Anton Sabashnikov, Nikhil P. Patil, Mohamed Zeriouh, Prashant N. Mohite, Bartlomiej Zych, Diana Garcia Saez, Bastian Schmack, Arjang Ruhparwar, Pascal M. Dohmen, Matthias Karck, Andre R. Simon, Alexander Weymann

Med Sci Monit Basic Res 2015; 21:9-14

DOI: 10.12659/MSMBR.893674