[Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin].
OBJECTIVE
To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line (HaCaT).


METHODS
The three pHSER-CCL20-shRNA-GFP vectors (pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control) have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lentiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively.


RESULTS
The titers of three lentiviruses were 7.08 x 10(5) transduced units (TU)/ml, 1.88 x 10(5) TU /ml and 2.08 x 10(5) TU/ml, respectively. Two HaCaT cell clones from each lentiviral vectors were obtained after G418 screening for 5 - 8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly.


CONCLUSIONS
The four human immortal keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.
Wang
Li-hua
LH
State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Peng
Dai-zhi
DZ
Liu
Jing
J
Zhou
Xin
X
Wang
Yong
Y
He
Sheng-dong
SD
He
Bin
B
Zheng
Bi-xiang
BX
Dong
Zheng-xue
ZX
Zhou
Guang-qian
GQ
D002460:Cell Line; D054418:Chemokine CCL20 / Q000235:genetics*; D002999:Clone Cells; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017768:Skin, Artificial; D023822:Tissue Engineering; D014162:Transfection
D054418:Chemokine CCL20; D034741:RNA, Small Interfering