One reason could be that your gene product is toxic to the cells.

We recommend testing the construct for expression of the gene of interest by transient Nucleofection® of your cells. If the proportion of expressing cells drops between 4 and 48 hours post Nucleofection®, this can be an indication of your gene product being toxic.

A different reason could be that the DNA is degraded. (Linearized) plasmid should always be checked on an agarose gel for integrity before Nucleofection®.