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

Human artificial chromosomes (HACs) were generated by transfer of
telomerized PAC constructs containing alpha satellite DNA of
various human chromosomes. To monitor which cells took up
constructs and subsequently formed stable clones under blasticidin
S (BS) selection, a CMV/EGFP expression cassette was inserted into
a HAC construct based on chromosome 5 alpha satellite DNA (142 kb).
Lipofection into HT1080 cells resulted in a small proportion of
cells exhibiting bright green fluorescence on day 1. Areas
containing such early green cells were marked, and plates monitored
over 2 weeks. In only one out of 41 marked areas, a viable clone
developed. In the remaining 40 areas, the green cells ceased
division at 1-8 cells. In contrast, outside the marked areas, 16
stable clones formed which did not exhibit green fluorescence
during the first cell divisions, but all cells of each became green
around day 4 -6. Fluorescence in situ hybridization (FISH) analysis
of isolated clonal lines demonstrated low copy HAC formation
without integration. 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. Copyright (C) 2004 S. Karger AG, Basel.