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.