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

For the development of human artificial chromosomes (HACs) as a gene transfer vehicle we need to assess the efficiency of de novo chromosome formation depending on the type and the copy number of transferred DNA constructs. In order to check transient EGFP expression as a reporter to immediately detect presence of transfected DNA, we microinjected approximately 1 to 10(5) copies of pEGFP-N1 plasmid into the nucleus of various cell types. Whether using primary, immortalized, or tumor cells, at least 10(3)-10(4) copies were required to generate a visible green signal in the majority of the 50-90% of cells surviving injection. Generally, the cells showed relatively constant, copy number-dependent signals. 10(3) copies resulted in faint and 10(5) in bright fluorescence under the microscope. In addition, the different copy number groups contained a small fraction of cells showing much stronger fluorescence, indicating activation or lack of suppression which facilitates detection of as few as 10(2) transferred copies in rare instances. Thus, transient expression from single copies is not sufficient to reliably detect presence of DNA in the nucleus. The result is relevant for the development of low copy HAC transfer protocols.