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For traditional cloning into pSilencer vectors, two DNA oligonucleotides that encode the chosen siRNA sequence are designed for insertion into the vector (Figures 2 and 3). In general, the DNA oligonucleotides consist of a 19-nucleotide sense siRNA sequence linked to its reverse complementary antisense siRNA sequence by a short spacer. Ambion scientists have successfully used a 9-nucleotide spacer (TTCAAGAGA), although other spacers can be designed. 5-6 T's are added to the 3' end of the oligonucleotide. In addition, for cloning into the pSilencer 1.0-U6 vector, nucleotide overhangs to the EcoR I and Apa I restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 2). In contrast, for cloning into the pSilencer 2.0-U6, 2.1-U6, 3.0-H1, or 3.1-H1 vectors, nucleotide overhangs with BamH I and Hind III restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 3). The resulting RNA transcript is expected to fold back and form a stem-loop structure comprising a 19 bp stem and 9 nt loop with 2-3 U's at the 3' end (Figure 1).
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Figure 2. Insert Design for pSilencer 1.0-U6. This insert is specific for the pSilencer 1.0-U6 Vector and contains the appropriate 3' overhangs for directional cloning into this vector. The loop sequence and length can be varied as desired.
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Figure 3 . Insert Design for pSilencer 2.0-U6 and pSilencer 3.0-H1. The insert design is specific for the pSilencer 2.0-U6, 2.1-U6, 3.0-H1 and 3.1-H1 Expression Vectors and contains the appropriate overhanging 5' ends for directional cloning into these plasmids. As with pSilencer 1.0-U6 shown in Figure 2, early indications suggest that a great deal of latitude is possible in the design of the loop; here we provide one loop sequence that we find works well.
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For cloning into the pSilencer adeno 1.0-CMV vector, DNA oligonucleotides with stem-loop structures are created similar to those of pSilencer 2.0 and 3.0 vectors described above. However, one notable exception is the absence of 5-6 T's from the 3'-end of the oligonucleotides for the CMV-based vector system since the transcription termination signal for the CMV-based vector system is provided by the SV40 polyA terminator. In addition, for cloning into the pSilencer adeno 1.0-CMV vector, nucleotide overhangs containing the Xho I and Spe I restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 4). However, for cloning into the pSilencer 4.1-CMV vector, nucleotide overhangs containing the Bam H1 and Hind III restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 5).
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Figure 4. Insert Design for pSilencer™ adeno 1.0-CMV Vector. This insert design is specific for the pSilencer adeno 1.0-CMV vector and contains the appropriate overhangs for directional cloning into this vector. The loop sequence and length can be varied as desired.
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