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Article: Targeted correction of a chromosomal point mutation by modified single-stranded oligonucleotides in a GFP recovery system

TitleTargeted correction of a chromosomal point mutation by modified single-stranded oligonucleotides in a GFP recovery system
Authors
KeywordsChimeraplasty
EGFP reporter system
Mutant enhanced green fluorescence protein gene (mEGFP)
RNA/DNA oligonucleotide
Single-stranded oligonucleotide
Strand bias
Targeted gene correction
Transfection
Issue Date2005
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/wps/find/journaldescription.cws_home/622790/description
Citation
Biochemical And Biophysical Research Communications, 2005, v. 334 n. 4, p. 1032-1041 How to Cite?
AbstractSynthetic oligonucleotides had been employed in DNA repair and promised great potentials in gene therapy. To test the ability of single-stranded oligonucleotide (SSO)-mediated gene repair within a chromosomal site in human cells, a HeLa cell line stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome was established. Transfection with specific SSOs successfully repaired the mEGFP gene and resulted in the expression of functional fluorescence proteins, which could be detected by fluorescence microscopy and FACS assay. Western blot showed that EGFP was only present in the cells transfected with correction SSOs rather than the control SSOs. Furthermore, DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. Using this reporter system, we determined the optimal structure of SSO by investigating the effect of length, modifications, and polarities of SSOs as well as the positions of the mismatch-forming nucleotide on the efficiency of SSO-mediated gene repair. Interestingly, we found that SSOs with mismatch-forming nucleotide positioned at different positions have varying potencies that homology at the 5′-end of SSOs was more crucial for the SSO's activity. These results provided guidance for designing effective SSOs as tools for treating monogenic inherited diseases. © 2005 Elsevier Inc. All rights reserved.
Persistent Identifierhttp://hdl.handle.net/10722/68022
ISSN
2015 Impact Factor: 2.371
2015 SCImago Journal Rankings: 1.152
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorYin, WXen_HK
dc.contributor.authorWu, XSen_HK
dc.contributor.authorLiu, Gen_HK
dc.contributor.authorLi, ZHen_HK
dc.contributor.authorWatt, RMen_HK
dc.contributor.authorHuang, JDen_HK
dc.contributor.authorLiu, DPen_HK
dc.contributor.authorLiang, CCen_HK
dc.date.accessioned2010-09-06T06:00:35Z-
dc.date.available2010-09-06T06:00:35Z-
dc.date.issued2005en_HK
dc.identifier.citationBiochemical And Biophysical Research Communications, 2005, v. 334 n. 4, p. 1032-1041en_HK
dc.identifier.issn0006-291Xen_HK
dc.identifier.urihttp://hdl.handle.net/10722/68022-
dc.description.abstractSynthetic oligonucleotides had been employed in DNA repair and promised great potentials in gene therapy. To test the ability of single-stranded oligonucleotide (SSO)-mediated gene repair within a chromosomal site in human cells, a HeLa cell line stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome was established. Transfection with specific SSOs successfully repaired the mEGFP gene and resulted in the expression of functional fluorescence proteins, which could be detected by fluorescence microscopy and FACS assay. Western blot showed that EGFP was only present in the cells transfected with correction SSOs rather than the control SSOs. Furthermore, DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. Using this reporter system, we determined the optimal structure of SSO by investigating the effect of length, modifications, and polarities of SSOs as well as the positions of the mismatch-forming nucleotide on the efficiency of SSO-mediated gene repair. Interestingly, we found that SSOs with mismatch-forming nucleotide positioned at different positions have varying potencies that homology at the 5′-end of SSOs was more crucial for the SSO's activity. These results provided guidance for designing effective SSOs as tools for treating monogenic inherited diseases. © 2005 Elsevier Inc. All rights reserved.en_HK
dc.languageengen_HK
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/wps/find/journaldescription.cws_home/622790/descriptionen_HK
dc.relation.ispartofBiochemical and Biophysical Research Communicationsen_HK
dc.subjectChimeraplastyen_HK
dc.subjectEGFP reporter systemen_HK
dc.subjectMutant enhanced green fluorescence protein gene (mEGFP)en_HK
dc.subjectRNA/DNA oligonucleotideen_HK
dc.subjectSingle-stranded oligonucleotideen_HK
dc.subjectStrand biasen_HK
dc.subjectTargeted gene correctionen_HK
dc.subjectTransfectionen_HK
dc.subject.meshChromosome Aberrationsen_HK
dc.subject.meshElectroporationen_HK
dc.subject.meshGene Targeting - methodsen_HK
dc.subject.meshGene Therapy - methodsen_HK
dc.subject.meshGene Transfer Techniquesen_HK
dc.subject.meshGreen Fluorescent Proteins - geneticsen_HK
dc.subject.meshHeLa Cellsen_HK
dc.subject.meshHumansen_HK
dc.subject.meshOligonucleotides - administration & dosage - geneticsen_HK
dc.subject.meshPoint Mutation - geneticsen_HK
dc.subject.meshPolymorphism, Single Nucleotide - geneticsen_HK
dc.subject.meshTransfection - methodsen_HK
dc.titleTargeted correction of a chromosomal point mutation by modified single-stranded oligonucleotides in a GFP recovery systemen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0006-291X&volume=334&spage=1032&epage=1041&date=2005&atitle=Targeted+correction+of+a+chromosomal+point+mutation+by+modified+single-stranded+oligonucleotides+in+a+GFP+recovery+systemen_HK
dc.identifier.emailWatt, RM:rmwatt@hku.hken_HK
dc.identifier.emailHuang, JD:jdhuang@hkucc.hku.hken_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.identifier.authorityHuang, JD=rp00451en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1016/j.bbrc.2005.06.193en_HK
dc.identifier.pmid16039616-
dc.identifier.scopuseid_2-s2.0-23044473394en_HK
dc.identifier.hkuros114544en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-23044473394&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume334en_HK
dc.identifier.issue4en_HK
dc.identifier.spage1032en_HK
dc.identifier.epage1041en_HK
dc.identifier.isiWOS:000231190900009-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridYin, WX=8988889100en_HK
dc.identifier.scopusauthoridWu, XS=37059359400en_HK
dc.identifier.scopusauthoridLiu, G=35181615300en_HK
dc.identifier.scopusauthoridLi, ZH=7409083059en_HK
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.scopusauthoridHuang, JD=8108660600en_HK
dc.identifier.scopusauthoridLiu, DP=8047815300en_HK
dc.identifier.scopusauthoridLiang, CC=7403280685en_HK

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