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Validation of Gene Transfection/Transduction

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Figure 1

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 a host cell's genome. In order to validate that a certain gene was introduced 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 both of these techniques.

Recent reports1-3 demonstrate the generation of induced pluripotent stem cells by introducing several transcription factors, Oct4, Sox2, cMyc, Lin28 and Nanog. TaqMan® Protein Assays are the perfect tool to measure the level of Oct4, Sox2, Klf4, Lin28 and Nanog protein expression in the cells to other common techniques such as western blot and immunostaining and without having to fix or stain cells or run a gel. Human dermal fibroblasts were transduced with a vector expressing OCT4, SOX2, KLF4 and RFP (red fluorescent protein) (Figure 1). RFP expression can be observed 24 hours post transduction, but cannot be observed 4 days post transduction. In contrast, OCT4, SOX2, and KLF4 protein expression was observed 24 hours and 4 days post transduction using TaqMan® Protein Assays.

References
1. 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.
2. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920.
3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, and Yamanaka S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872.


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