Fast and efficient multitransgenic modification of human pluripotent stem cells.

Human gene therapy methods (2014-02-04)
Kristin Schwanke, Sylvia Merkert, Henning Kempf, Susann Hartung, Monica Jara-Avaca, Christian Templin, Gudrun Göhring, Axel Haverich, Ulrich Martin, Robert Zweigerdt
要旨

Human pluripotent stem cells (hPSCs) represent a prime cell source for pharmacological research and regenerative therapies because of their extensive expansion potential and their ability to differentiate into essentially all somatic lineages in vitro. Improved methods to stably introduce multiple transgenes into hPSCs will promote, for example, their preclinical testing by facilitating lineage differentiation and purification in vitro and the subsequent in vivo monitoring of respective progenies after their transplantation into relevant animal models. To date, the establishment of stable transgenic hPSC lines is still laborious and time-consuming. Current limitations include the low transfection efficiency of hPSCs via nonviral methods, the inefficient recovery of genetically engineered clones, and the silencing of transgene expression. Here we describe a fast, electroporation-based method for the generation of multitransgenic hPSC lines by overcoming the need for any preadaptation of conventional hPSC cultures to feeder-free conditions before genetic manipulation. We further show that the selection for a single antibiotic resistance marker encoded on one plasmid allowed for the stable genomic (co-)integration of up to two additional, independent expression plasmids. The method thereby enables the straightforward, nonviral generation of valuable multitransgenic hPSC lines in a single step. Practical applicability of the method is demonstrated for antibiotic-based lineage enrichment in vitro and for sodium iodide symporter transgene-based in situ cell imaging after intramyocardial cell infusion into explanted pig hearts.

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2-メルカプトエタノール, ≥99.0%
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2-メルカプトエタノール, for molecular biology, suitable for electrophoresis, suitable for cell culture, BioReagent, 99% (GC/titration)
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DAPI, for nucleic acid staining
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2-メルカプトエタノール, BioUltra, for molecular biology, ≥99.0% (GC)
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2-メルカプトエタノール, for HPLC derivatization, LiChropur, ≥99.0% (GC)
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2-メルカプトエタノール, ≥99.0%
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2-メルカプトエタノール, SAJ special grade, ≥99.0%
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抗SOX17抗体 ウサギ宿主抗体, affinity isolated antibody
Sigma-Aldrich
抗SOX17抗体 ウサギ宿主抗体, affinity isolated antibody

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