Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.

Shinta Saito,Noritaka Adachi,Kiyoe Ura,Miho Kodama

doi:10.1186/s13104-015-1241-6

Abstract

BackgroundTargeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process.ResultsBy virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors.ConclusionsOur methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1241-6) contains supplementary material, which is available to authorized users.

Highlights

Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals
To create entry clones for floxed promoterless markers, a hygromycin-resistance gene (HygR), a puromycinresistance gene (PuroR), a neomycin-resistance gene (NeoR), and a bifunctional lacZ/NeoR gene were each subcloned into pENTR lox71-P at the AscI and/or ClaI sites, with an IRES, IRES2 or 2A peptide sequence added upstream of each drug-resistance gene
PENTR lox71P IRES-Hyg was constructed by subcloning a 2.2-kb XhoI/NgoMIV fragment containing IRES, HygR and polyA sequences into ClaI-digested pENTR lox71-P. pENTR lox71P IRES-Puro was constructed by subcloning a 1.3-kb MluI/PvuI fragment containing IRES, PuroR and polyA sequences into AscI/ClaI-digested pENTR lox71-P. pENTR lox71P IRES-Neo was constructed by subcloning a 1.7-kb EcoRI/BamHI fragment containing IRES, NeoR and polyA sequences into ClaI-digested pENTR lox71-P. pENTR lox71P IRES2-βgeo was constructed with an In-Fusion®HD cloning kit (Clontech): a 4.7-kb fragment containing IRES2, βgeo and polyA sequences was PCR amplified with primers In-Fus Fw and In-Fus Rv, and the PCR fragment and ClaI-digested pENTR lox71-P were subjected to In-fusion cloning, yielding pENTR lox71P IRES2-βgeo

Summary

Introduction

Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. Gene targeting via homologous recombination provides a powerful means for studying gene function by a reverse genetic approach [1, 2] This technology depends on homologous recombination reactions that occur between transfected DNA (i.e., targeting vector) and the host genome [3, 4]. The presence of a toxic gene in the targeting vector is expected to kill random integrants (i.e., clones with off-target integration via non-homologous recombination), while having little impact on homologous recombination [10] It appears, that this strategy has a limited efficacy of counterselection (2–3-fold enrichment at the most) and is not routinely applicable to gene-targeting experiments

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Conclusion