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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
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 lines by jetPEI liposome were 75.1±3.4% and 1.3±0.2%, respectively. The transfection efficiency of ploxP-hCCL20-EGFP into HaCaT cells using nucleofection electroporation was 0.3±0.1% (P=0.000), but the positive control vector pmaxGFP (3,490 bp) using the same method was 38.3±2.8%.
Conclusions: Overall low transfection efficiencies of ploxP-hCCL20-EGFP into HaCaT cells, regardless of transfection method, may either be due to the high molecular weight of the vector or to the fact that this particular cell line may be inherently difficult to transfect.
Keywords: Chemokine CCL20 - genetics, Blotting, Southern, Cloning, Molecular, DNA Primers - genetics, Electroporation, Gene Knockout Techniques - methods, Gene Targeting - methods, Genetic Vectors - genetics, Keratinocytes, Microscopy, Fluorescence, Polymerase Chain Reaction, Statistics, Nonparametric, Transfection - methods