Abstract
BackgroundStable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters.ResultsWe quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order.ConclusionsThe results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy.
Highlights
Stable expression of transgenes is an important technique to analyze gene function
Kinetics of the drug-selection systems in the internal ribosome entry site (IRES)-based expression vectors To evaluate the kinetics of the bi-cistronic expression vectors carrying various drug resistance genes in mouse embryonic stem (mES) cells, we constructed a bi-cistronic expression vector system using enhanced green fluorescent protein (Egfp) as an indicator of the expression level and drug resistance genes under the control of the IRES from encephalomyocarditis virus (EMCV) driven by the CAG expression unit [10] (Figure 1A)
These evidences accorded to our experiences that zeo and pac efficiently worked to select mES cell lines expressing fluorescent markers ubiquitously and strongly in chimeric embryos [11], and that neo gave higher numbers of LIF-independent colonies than pac when applied to select drive the expression of Tbx3 transgene, of which the expression at high level is toxic in mES cells [12]
Summary
Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been used as selection markers to isolate a transfectant without considering their dose-dependent characters. Streptomyces alboniger (pac) against puromycin, hygromycin B phosphotransferase from Escherichia coli (hph) aginst hygromycin B, Streptoalloteichus hindustanus ble (Sh ble: designated as zeo in this paper) against the bleomycin derivative zeocin, blasticidin S deaminase from Aspergillus terreus (bsd) against blasticidin S, and histidinol dehydrogenase from Salmonella typhimurium (hisD) against histidinol [1,2,3,4,5,6]. These drugs and the resistance genes have been regarded as dominant selection markers that reflect the introduction of the transgenes into mammalian cells. In this strategy, the drug resistance does not always appropriately reflect the expression level of the transgene because generally the stable expression levels of exogenous expression cassettes are highly sensitive to thier sites of integration, as a result of the local chromatin environment when the transgenes are randomly integrated into the host genome [7], which affect the expression levels of the drug resistance gene cassette and the transgene cassette separately
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