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. 2012 Apr 20;7(4):e35619. doi: 10.1371/journal.pone.0035619

Figure 1. Transgene construct and cell line isolation and rederivation scheme.

Figure 1

(a) The transgene construct is comprised of a puromycin-resistance gene (Puro) and an EGFP gene (EGFP), each driven by an individual TK promoter (pTK) and flanked firstly by a pair of incompatible lox sites (loxP, lox2272) and secondly homologous sequence arms of the bovine beta-casein promoter (5′ β-CN) and 3′ untranslated region (3′ β-CN). (b) Primary bovine cells were transfected and cell clones isolated following antibiotic selection. Two cell clones (A and B) were chosen as source for donor cells to generate embryos using SCNT. Following transfer into recipient cows for further in vivo development, two resulting fetuses generated from cell line A (fetus A1, A2) and cell line B (fetus B1 and B2) were used to isolate a series of rejuvenated primary cell lines with the same genetics as the original cell lines A (A1-1, A1-2, A2-3, A2-4, A2-5, A2-6) and B (B1-1, B1-2, B2-1).