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Mechanistic basis for increased human gene targeting by promoterless vectors-roles of homology arms and Rad54 paralogs
- Source :
- The FEBS journal. 284(17)
- Publication Year :
- 2017
-
Abstract
- Gene targeting by homologous recombination provides the definitive tool for analyzing gene function. Promoterless vectors, which do not possess a promoter to drive marker gene expression, confer higher targeting efficiencies than conventional vectors due to the reduced number of drug-resistant clones. We here show that gene-targeting efficiency is typically ≥ 25% with the use of exon-trapping-type promoterless vectors in a human diploid cell line, Nalm-6. The efficiency of exon-trapping gene targeting was correlated with the level of target gene expression when a 2A peptide sequence was linked to the marker gene. Intriguingly, total arm length was not necessarily a determinant of targeting efficiency, as longer arms tend to enhance both homologous (targeted) and nonhomologous (nontargeted) integration of the vector; rather, the presence of an exon in the 5' arm led to a decreased targeting efficiency. Strikingly, loss of Rad54 did not severely affect the targeting efficiency of exon-trap vectors. Moreover, additional deletion of the Rad54 paralog Rad54B had limited impact on the high-efficiency gene targeting. These results indicate that targeted integration occurs in human cells even when both Rad54 and Rad54B are missing. These studies provide additional important insight into the contribution of various DNA repair factors on the targeting mechanics.
- Subjects :
- 0301 basic medicine
DNA repair
Genetic Vectors
Biology
Biochemistry
Marker gene
Homology (biology)
Cell Line
03 medical and health sciences
Exon
Gene Knockout Techniques
Humans
Site-specific recombinase technology
Promoter Regions, Genetic
Molecular Biology
Gene
Genetics
DNA Helicases
Gene targeting
Nuclear Proteins
Cell Biology
Exons
DNA-Binding Proteins
030104 developmental biology
Genetic Loci
Homologous recombination
Subjects
Details
- ISSN :
- 17424658
- Volume :
- 284
- Issue :
- 17
- Database :
- OpenAIRE
- Journal :
- The FEBS journal
- Accession number :
- edsair.doi.dedup.....4a044fdc9c42c2fe086734aebd3bccbc