Validation of Gene Transfection / Transduction
Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector. These are common tools to introduce a foreign gene into host cells. A common way to validate that a genetic material was successfully introduced into cells is to measure protein expression. This is typically performed by Western blot or immunostaining, however, TaqMan® Protein Assays offer a faster, more sensitive and more quantitative alternative to these techniques.
Recent reports (1–3) demonstrate the generation of induced pluripotent stem cells by introducing the transcription factors Oct4, Sox2, cMyc, Lin28 and Nanog. TaqMan® Protein Assays are the perfect tool to measure the levels of Oct4, Sox2, Klf4, Lin28 and Nanog protein expression in cells. Unlike common protein analysis techniques, such as western blot and immunostaining, with TaqMan® Protein Assays, there is no need to fix or stain cells, or run and transfer gels.
TaqMan® Protein Assays Offer a Faster, More Sensitive & Quantitative Alternative to Western Blot or Immunostaining
In this experiment, human dermal fibroblasts were transduced with a vector expressing OCT4, SOX2, KLF4 and RFP (red fluorescent protein). RFP expression can be observed 24 hours after transduction, but is no longer detectable 4 days after transduction by fluorescence microscopy. In contrast, OCT4, SOX2, and KLF4 protein expression was observed at both 24 hours and 4 days post transduction using TaqMan® Protein Assays.
- Takahashi K, and Yamanaka S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676.
- Yu J et. al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920.
- Takahashi K et. al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872.
- Ruff D. (2011) Applications of Quantitative PCR Protein Assays During Reprogramming. Stem Cells Dev. 2011 Apr 8. [Epub ahead of print] PMID: 21476854.