Table 2.
Oligos used for generating shRNA constructs by PCR and transfected into amebae
| Oligo Name | Oligo Sequence |
|---|---|
| U6 HindIII forward | CTACTGAAGCTTGTTTTTATGAAAAAGTGTATTTGC |
| GFP R1 | TCTCTTGAAGTTTTCCGTATGTTGCATCACCTTGGGCCCAATTTTATTTTTCTTTTTATCC |
| GFP R2 | TCGATCGCGGCCGCAAAAAAGGTGATGCAACATACGGAAAACTCTCTTGAA |
| Igl1 (272–300) R1 | TCTCTTGAAATTTCCAGAGTGTGATGATGTATTTACTTGGGCCCAATTTTATTTTTCTTTTTATCC |
| Igl1 (272–300) R2 | TCGATCGCGGCCGCAAAAAAGTAAATACATCATCACACTCTGGAAATTCTCTTGAA |
| Igl (1198–1226) R1 | TCTCTTGAACAATGAGTTCCATTCAATGTAAGTCCATTGGGCCCAATTTTATTTTTCTTTTTATCC |
| Igl (1198–1226) R2 | TCGATCGCGGCCGCAAAAAATGGACTTACATTGAATGGAACTCATTGTCTCTTGAA |
| Igl (2412–2440) R1 | TCTCTTGAAGTCCACTAAAACCATCTGAACATTCTGTTGGGCCCAATTTTATTTTTCTTTTTATCC |
| Igl (2412–2440) R2 | TCGATCGCGGCCGCAAAAAACAGAATGTTCAGATGGTTTTAGTGGACTCTCTTGAA |
| Igl (2777–2805) R1 | TCTCTTGAATGGTGATGTGCATGGTATACATGTTCCTTGGGCCCAATTTTATTTTTCTTTTTATCC |
| Igl (2777–2805) R2 | TCGATCGCGGCCGCAAAAAAGGAACATGTATACCATGCACATCACCATCTCTTGAA |
| URE3-BP (350–378) R1 | TCTCTTGAAGTTCATAACGAAGAGATTGTATGCAAGTTGGGCCCAATTTTATTTTTCTTTTTATCC |
| URE3-BP (350–378) R2 | TCGATCGCGGCCGCAAAAAACTTGCATACAATCTCTTCGTTATGAACTCTCTTGAA |
| URE3-BP (580–608) R1 | TCTCTTGAAAATGGTTTCATTGGACCATAGTATGGATTGGGCCCAATTTTATTTTTCTTTTTATCC |
| URE3-BP (580–608) R2 | TCGATCGCGGCCGCAAAAAATCCATACTATGGTCCAATGAAACCATTTCTCTTGAA |
| EhC2A (363–391) R1 | TCTCTTGAATCATGCCTGGTTGCATTGGTGGAACCATTGGGCCCAATTTTATTTTTCTTTTTATCC |
| EhC2A (363–391) R2 | TCGATCGCGGCCGCAAAAAATGGTTCCACCAATGCAACCAGGCATGATCTCTTGAA |
| EhC2A (502–530) R1 | TCTCTTGAAATTGGTGGATATCCAGGTGGTGGGTAAGCGGGCCCAATTTTATTTTTCTTTTTATCC |
| EhC2A (502–530) R2 | TCGATCGCGGCCGCAAAAAAGCTTACCCACCACCTGGATATCCACCAATTCTCTTGAA |
| EhC2A (363–391 scrambled) R1 | TCTCTTGAAATCTGGAACGGTCTGGATTGTCTAGCCTTGGGCCCAATTTTATTTTTCTTTTTATCC |
| EhC2A (363–391 scrambled) R2 | TCGATCGCGGCCGCAAAAAAGGCTAGACAATCCAGACCGTTCCAGATTCTCTTGAA |
The sequences shown in Table 1 were used to design primers for two-step PCR, based on the method used by Gou et al (2003) [30] and diagrammed in Figure 1A. The final PCR product contained the E. histolytica U6 promoter with a HindIII site on the 5' end, an ApaI site at the 3' end of the U6 promoter, the 29-nt sense strand of the hairpin, the 9 bp loop TTCAAGAGA, the antisense strand of the hairpin, and the U6 terminator sequence followed by a NotI restriction site. The forward primer, "U6 HindIII forward", contained the HindIII recognition site and the 5' end of the U6 promoter, the first reverse primer (R1) contained the sequence of the sense strand of the shRNA and the future loop, and the second reverse primer (R2) contained the loop sequence, the antisense strand sequence, and the U6 termination sequence. A control GFP sequence [30] was used to design oligos for creating a shRNA construct as a transfection control.