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A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis

Nature Protocols volume 3, pages 1550–1558 (2008)Cite this article

Abstract

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take ∼1 month.

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Figure 1: Morphology of human embryonic stem cells (HESCs) prepared for transfection of the pEFBOS-CreIRESpuro vector by FuGENE 6 Transfection Reagent and following selection in puromycin.
Figure 2: Photomicrographs of human embryonic stem cells (HESCs) cloned by single-cell deposition.
Figure 3: Possible primer locations for human embryonic stem cell (HESC) colony screening for excision of the positive selection cassette.

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Acknowledgements

We thank Robyn Mayberry, Kathy Koutsis and Amanda Bruce for the provision of HESCs. This work was supported by the Australian Stem Cell Centre, the National Health and Medical Research Council of Australia (NHMRC) and the Juvenile Diabetes Research Foundation. A.G.E. is a senior research fellow of the NHMRC.

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  1. Monash Immunology and Stem Cell Laboratories, STRIP 1, Building 75, Level 3, Monash University, Clayton, 3800, Victoria, Australia

    Richard P Davis, Magdaline Costa, Catarina Grandela, Andrew M Holland, Tanya Hatzistavrou, Suzanne J Micallef, Xueling Li, Adam L Goulburn, Lisa Azzola, Andrew G Elefanty & Edouard G Stanley

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  1. Richard P Davis
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  2. Magdaline Costa
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Correspondence to Edouard G Stanley.

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Davis, R., Costa, M., Grandela, C. et al. A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis. Nat Protoc 3, 1550–1558 (2008). https://doi.org/10.1038/nprot.2008.146

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