TRANSIENT TRANSFECTION OF THE ENTERIC PARASITE ENTAMOEBA-HISTOLYTICA AND EXPRESSION OF FIREFLY LUCIFERASE

Development of DNA-mediated transfection in Entamoeba histolytica will facilitate basic research toward the control of this protozoan parasi te. A transient transfection system was established by using the firef ly luciferase gene ligated to the 5' and 3' flanking regions of the am ebic hgl1 gene. The optimal construct tested encoded ad hgl1-luciferas e fusion protein and contained 1 kb of 5' flanking sequence with 16 ba ses of coding sequence from the hgl1 gene ligated in frame to the luci ferase start codon and 2.3 kb of 3' flanking sequence from hgl1 ligate d 3' to the luciferase stop codon. Optimal electroporation conditions in strain HM-1:IMSS trophozoites when using this construct were 500 mu F and 500 V/cm, which resulted in luciferase activity up to 5000-fold above back ground 9-12 hr after electroporation. Constructs that cont ained the luciferase gene without amebic flanking sequences or that co ntained a simian virus 40 promoter, enhancer, and polyadenylylation Si gnal produced only background levels of luciferase activity. The abili ty to introduce and express genes in amebae will now permit a genetic analysis of the virulence of this organism, which remains a serious th reat to world health.