Integrated NeoGGTE1bGFP sequences in stable cell lines.

Abstract

<p>DNA was extracted from G418-resistant stable cell lines established with the plasmid pNeoGGTE1bGFP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#pone-0015704-g001" target="_blank">Fig. 1b</a>). Blots were prepared and probed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#s4" target="_blank">Materials and Methods</a>. Lane designations: M, size markers derived from an <i>Eco</i>RI/<i>Psh</i>A1 digest of the plasmid (sizes shown on the left); 2, reconstruction with 2 copies of <i>Sph</i>I-digested (linear) plasmid (7.2 kbp), based on quasi-tetraploid human DNA content; 20, reconstruction with 20 copies of plasmid; A+, GFP-expressing cell line 1 isolated after electroporation of 1 µg of DNA; A−, GFP-negative cell line from the same experiment as A+; B+, GFP-expressing cell line isolated in a second experiment after electroporation of 1 µg of DNA; B−, GFP-negative cell line from the same experiment as B+; C+, GFP-expressing cell line isolated after lipofection with 1 µg of DNA; C−, GFP-negative cell line 2 from the same experiment as C+. An irrelevant lane was removed from between lanes 20 and A+. The diagram below shows the results expected from a single (top) or tandem (bottom) integration of the complete plasmid sequence (open box). For the former, the sizes of the two junction DNA fragments that hybridize to the probe (shaded box) will depend on the site of insertion. For the latter, in addition to the junction fragments, a unit length band (7.2 kbp) will be generated and its intensity will depend on the number of copies in the tandem array. There is a single site for <i>Sph</i>I (S) cleavage in the plasmid <i>neo</i> gene.</p