An Alternate Approach to Generate Induced Pluripotent Stem Cells with Precise CRISPR/Cas9 Tool

Figure 2

Knock-in and validation of reprogramming donor cassette. (a) Design of the DOX-inducible polycistronic expression cassette of reprogramming factors OCT4, SOX2, and KLF4. Each component is named and labeled with a different color. The red arrows under the cassette represent the restriction sites, and the cassette is flanked by sequences homologous to CASH-1. The DNA sequence of the whole cassette is shown in supplementary figure 3. (b) Detection of a GFP signal in donor-transfected HEK293T cells with or without DOX induction. Scale bar: 500 μm. (c) Confirmation of protein expression of the reprogramming factors in the donor-transfected HEK293T cells with or without DOX induction. (d) Tabular plan followed for optimization of the donor cassette knock-in in HEK293T cells. (e and f) The 5 (e) and 3 (f) junction PCR assays of the template genomic DNA isolated from selected HEK293T colonies. (g) Tabular plan followed for optimizing the donor cassette knock-in in HDFs. (h) 5 and 3 junction PCR assays of the template genomic DNA isolated from selected HDF colonies. (i and j) Confirmation of mRNA expression of each reprogramming factor in HEK293T-OSK (i) and HDF-OSK cells (j) in comparison with respective normal cells (WT), where GAPDH mRNA served as a loading control. (k) Confirmation of GFP expression in HDF-OSK cells with or without DOX induction for 24 h under fluorescence microscope. Scale bar: 200 μm. L: 1-kbp ladder.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)