SpringerProtocols: Abstract: Transfection and Promoter Analysis in Embryonic Stem Cells

Transfection and Promoter Analysis in Embryonic Stem Cells

By: Sangmi Chung², Kwang-Soo Kim²

Abstract

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Embryonic stem (ES) cells are useful for the molecular analysis of developmental pathways when combined with efficient genetic manipulation and in vitro differentiation procedures. To facilitate the genetic modification of ES cells, we describe how to analyze the transcriptional activities of different promoter systems. The DNA segments from a number of promoters are subcloned into the multiple cloning sites of the green fluorescent protein (GFP) reporter plasmid. The resulting recombinant plasmids are used to transfect ES cells by lipofection. Using luciferase-normalized GFP assays, we observed differing promoter activities in transient transfections of ES cells. The judicious selection of the optimum promoter system for specific genetic modification of ES cells will ensure the successful generation of transgenic ES cell lines.

Affiliation(s): (2) Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA

Book Title: Embryonic Stem Cell Protocols: Volume 1: Isolation and Characterization

Series: Methods in Molecular Biology | Volume: 329 | Pub. Date: Feb-15-2006 | Page Range: 187-193 | DOI: 10.1385/1-59745-037-5:187

Subject: Cell Biology

Key Words: Embryonic stem cells - GFP assay - luciferase assay - promoter strength - transfection - transgene expression

References

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