Transient transfection of the enteric parasite Entamoeba histolytica and expression of firefly luciferase.

Abstract

Fig. 1), the 3' portion of the luciferase gene from pGEM-luc was amplified by using the polymerase chain reaction (PCR) with the primers 94 and 95 (all nucleotides used for plasmid construction are described in Table 1), which added a syntheticXho I site in the amplified product two bases 3' of the stop codon of luciferase. The amplified product and pGEM-luc were digested with Cla I and Xho I and were ligated together with T4 DNA ligase (GIBCO/BRL). By effectively deleting 53 bases between the stop codon of the luciferase gene and the Xho I site in the multicloting site ofpGEM-luc, 3' amebic sequences could be ligated in close proximity to the 3' terminus of the reporter gene. A short 3' untranslated region in amebic mRNA is typical and may prove critical to message stability.To make the AA2R8 construct, approximately 1 kb of the 5' flanking region of hgll was PCR-amplified from a genomic clone containing the 5' coding region and flanking region of hgll (6) Abbreviations: E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; E-64c, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane; SV40, simian virus 40. *To whom reprint requests should be addressed. 7099The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

“…Currently, we are using this new technology to study organelle biogenesis, drug-resistance and gene transcription. These transformation assays should provide invaluable tools for advancing our knowledge of the basic biology and pathogenesis of trichomonads, as similar methods developed in the past few years for other protozoan parasites (19)(20)(21)(22)(23)(24)(25)(26)(27)(28) have done. FIG.…”

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

“…To name but a few, the signal for the initiation of the invasion process, the mechanism of elimination of the mucus barrier, the role of EDG, and the in vivo participation of the different molecules in this multifactorial process have not been demonstrated. The recent advances in amebic transfection technology (56,98,104,106,122,168) will undoubtedly shed new light on the involvement of specific molecules in the pathogenic mechanism. The uncertainty of the ploidy of E. histolytica has hindered the production of gene knockout clones like those produced for the study of the pathogenicity of Toxoplasma gondii and Leishmania spp.…”

Identification and characterization of a cyclooxygenase-like enzyme fromEntamoeba histolytica

“…The COX-like sequence was subcloned at HindIII and BamHI sites in pHTP-luc expression vector and designated HTP-COX. This construct was used to transfect E. histolytica to overexpress the COX-like enzyme as described (30). After transfection, amebae were allowed to grow for 24 h without antibiotic selection in TYI-S-33 medium and then selected against 6 g͞ml G418.…”