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Article: Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin

TitleClone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin
Authors
Issue Date2009
Citation
Zhonghua Wai Ke Za Zhi [Chinese Journal Of Surgery], 2009, v. 47 n. 8, p. 621-624 How to Cite?
AbstractOBJECTIVE: 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.
Persistent Identifierhttp://hdl.handle.net/10722/170162
ISSN
2015 SCImago Journal Rankings: 0.133

 

DC FieldValueLanguage
dc.contributor.authorWang, LHen_US
dc.contributor.authorPeng, DZen_US
dc.contributor.authorLiu, Jen_US
dc.contributor.authorZhou, Xen_US
dc.contributor.authorWang, Yen_US
dc.contributor.authorHe, SDen_US
dc.contributor.authorHe, Ben_US
dc.contributor.authorZheng, BXen_US
dc.contributor.authorDong, ZXen_US
dc.contributor.authorZhou, GQen_US
dc.date.accessioned2012-10-30T06:05:43Z-
dc.date.available2012-10-30T06:05:43Z-
dc.date.issued2009en_US
dc.identifier.citationZhonghua Wai Ke Za Zhi [Chinese Journal Of Surgery], 2009, v. 47 n. 8, p. 621-624en_US
dc.identifier.issn0529-5815en_US
dc.identifier.urihttp://hdl.handle.net/10722/170162-
dc.description.abstractOBJECTIVE: 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.en_US
dc.languageengen_US
dc.relation.ispartofZhonghua wai ke za zhi [Chinese journal of surgery]en_US
dc.subject.meshCell Lineen_US
dc.subject.meshChemokine Ccl20 - Geneticsen_US
dc.subject.meshClone Cellsen_US
dc.subject.meshGene Knockdown Techniquesen_US
dc.subject.meshGenetic Vectorsen_US
dc.subject.meshHumansen_US
dc.subject.meshLentivirus - Geneticsen_US
dc.subject.meshRna, Small Interfering - Geneticsen_US
dc.subject.meshSkin, Artificialen_US
dc.subject.meshTissue Engineeringen_US
dc.subject.meshTransfectionen_US
dc.titleClone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skinen_US
dc.typeArticleen_US
dc.identifier.emailZhou, GQ:wormoscz@gmail.comen_US
dc.identifier.authorityZhou, GQ=rp00527en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid19595046-
dc.identifier.scopuseid_2-s2.0-77955477232en_US
dc.identifier.volume47en_US
dc.identifier.issue8en_US
dc.identifier.spage621en_US
dc.identifier.epage624en_US
dc.identifier.scopusauthoridWang, LH=8912385300en_US
dc.identifier.scopusauthoridPeng, DZ=8600624500en_US
dc.identifier.scopusauthoridLiu, J=26657479700en_US
dc.identifier.scopusauthoridZhou, X=7410091709en_US
dc.identifier.scopusauthoridWang, Y=14038395700en_US
dc.identifier.scopusauthoridHe, SD=35332018500en_US
dc.identifier.scopusauthoridHe, B=7402047143en_US
dc.identifier.scopusauthoridZheng, BX=27468148600en_US
dc.identifier.scopusauthoridDong, ZX=9040125500en_US
dc.identifier.scopusauthoridZhou, GQ=23394245100en_US

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