Construction of Versatile Shuttle Vectors

The pEhActNeo shuttle vector, which served as the basic construct, contains the Neo gene, which confers resistance to G418, flanked by the 5′ and 3′ regulatory sequences of the amoeba actin 1 gene Ehactin [56 (link)–58 ] and the E. histolytica autonomous replication sequence, both cloned in pBluescript II SK (−). Primers used for the construction of the different cassettes are listed in Table 2.
Plasmid pB33 includes the 5′ upstream segment (473 bp) of the Ehap-a gene amplified by PCR from psAP-1 [12 (link)] using primers A and B (Table 2). The ORF region of the Ehlgl1 gene and the 3′ Ehactin flanking region were prepared by digestion of the intermediate plasmid construct pSA21 with NcoI/BamHI [35 (link),47 ]. This plasmid is based on pBluescript IIKS and includes a cassette containing the 5′ flanking region of the Ehactin gene, the ORF of the Ehlgl1 (867bp) gene, and the 3′ flanking region of Ehactin.
To construct plasmid pAY we first generated, by PCR with plasmid psAP-1 [12 (link)] as a template and primers A and C (Table 2), a 521 bp fragment that included the 473 bp of the 5′ upstream region of the Ehap-a gene and 44 bp of the signal peptide of the Ehap-a gene. This fragment was then ligated to the segment containing the ORF of Ehlgl1 with the 3′ Ehactin flanking region as above.
The construction of pAP-CP5 was similar to that of pB33. The 473 bp 5′ upstream of the Ehap-a gene was generated by PCR using primers A and B (Table 2) on plasmid psAP-1 [12 (link)]. The ORF of EhCP-5 [41 (link),42 (link)] was generated by PCR using primers D and E (Table 2) on genomic DNA of strain HM-1:IMSS, and the 3′ flanking region of Ehactin was generated by digestion of plasmid pSA21 [47 ] with SalI/BamHI.
For plasmid pSG, a DNA fragment containing 188 bp of the 5′ upstream region of Ehap-a consisting of the truncated SINE1 sequence and the T-rich region was amplified by PCR from plasmid psAP-1 [12 (link)] using primers A and F (Table 2). The resulting 188 bp fragment was ligated to the 5′ end of another segment (1,203 bp) consisting of the Ehlgl1 ORF and 338 bp of the Ehlgl1 5′ upstream region. This latter fragment was generated by PCR amplification of genomic DNA of strain HM-1:IMSS using primers G and H (Table 2) followed by ligation to the 3′ Ehactin regulatory segment.
Plasmid pP10 was constructed from a DNA fragment of 275 bp of the 5′ upstream region of the Ehap-a gene generated by PCR from plasmid psAP-1 [12 (link)] using primers I and B (Table 2) and ligated to the ORF of Ehlgl1 with the 3′ actin flanking region, as in pB33.
For plasmid pTL, the 473 bp fragment of the 5′ upstream region of Ehap-a and a fragment of 421 bp starting from the 5′ end of the ORF of Ehlgl1 (truncated Ehlgl1) (894 bp) were generated by PCR from plasmid pB33 using primers A and K (Table 2)
Plasmid pSV contains a 338 bp fragment from the 5′ upstream region of Ehlgl1 generated by PCR from genomic DNA using primers J and G. This fragment was ligated to the ORF of the EhCP-5 gene and then to the 3′ flanking region of the Ehactin gene.
Each of the cassettes mentioned above was then ligated to the digested and dephosphorylated pEhActNeo shuttle vector as previously described [12 (link),21 (link)]