Visible transient expression of EGFP requires intranuclear injection of large copy numbers

Suitability of a CMV/EGFP cassette to monitor stable expression from human artificial chromosomes but not transient transfer in the cells forming viable clones

“…Consistently, copy numbers of 10 3 per nucleus were required in various cell types including HT1080, and 10 4 or 10 5 copies led to brighter signals, detectable within 2 h post injection. However, in a small fraction of F5 % of microinjected cells, exceptionally high expression occurred, which allowed the detection of as few as 10-100 copies (Schindelhauer and Laner, 2002). According to these experiments, it is obvious that the number of cells showing transient expression can be highly variable (0-100 %), even if 100 % of cells physically obtained a copy number somewhere between 1 and 10 3 .…”

“…However, it could be that the 41 early green cells belonged to a much larger fraction with moderate copy numbers, out of which only a small set was detectable due to exceptionally high expression (according to 10-100 plasmid copies being detectable in F5% of cells in a microinjection, see Schindelhauer and Laner, 2002). In this case, a large number of physically transfected cells would not be detected by transient expression.…”

“…The specific question was whether transient expression from HAC constructs, visible under the microscope one day after lipofection, was a measure of the number of physically successfully transfected cells, and how many of these cells would form stable clones. We questioned this, because we recently found that a high copy number (1 10 3 ) of the CMV/EGFP expression cassette present on linear or circular plasmid DNA (pEGFP-N1, Clontech) is required to detect green fluorescence at day 1 in the majority of cells (HT1080 and other cell types), which physically obtained the DNA by intranuclear microinjection (Schindelhauer and Laner, 2002). Transfection of HAC constructs in this copy number range (10 3 per cell) would require transfer of a large amount of DNA, since the 1.7-kb-sized CMV/EGFP/ SV40-polyA expression cassette occupies only 1.2 % of the HAC construct, compared to 36 % of the EGFP-N1 plasmid.…”

“…1b). Function of the EGFP cassette within the sequence context of vector pTT was checked using intranuclear microinjections (10 4 -10 5 copies per injection volume) and transient expression analysis according to the assay previously described (Schindelhauer and Laner, 2002).…”

“…We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation. …”