Note: The vector sequence file has been compiled from information in the sequence database, published literature, and other sources, together with partial sequences obtained by BD Biosciences Clontech. This vector has not been completely sequenced.

Description

The RNAi-Ready pSIREN-DNR Donor Vector, a component in the BD Knockout? Adenoviral System 2 (Cat. No. 631529), is designed to express a small interfering ds RNA (siRNA) driven by the human U6 promoter (PU6; RNA Pol III-dependent). RNAi-Ready pSIREN-DNR is provided as a linearized vector digested with BamH I and EcoR I. It is used for targeted gene silencing when an oligonuceotide encoding an appropriate siRNA is ligated into the vector. pSIREN-DNR has been designed as a Donor Vector to transfer the siRNA cassette to the adenoviral Acceptor Vector pLP-Adeno-X-PRLS Viral DNA by Cre/loxP mediated recombination. In addition, recombinant pSIREN-DNR can be used directly in transient transfection experiments to screen siRNA constructs for functionality.

Cre, a 38-kDa recombinase protein from bacteriophage P1, mediates recombination between DNA sequences at specific locations called loxP sites (1, 2). The pSIREN-DNR Vector contains two loxP sites, which flank the 5' end of the siRNA expression cassette and the 5' end of the open reading frame encoding the chloramphenicol resistance gene (Cmr). Once your oligonucleotide is inserted, the resulting Donor Vector construct can be combined with any promoterless Acceptor Vector and Cre recombinase; Cre mediates the transfer of the siRNA cassette to the Acceptor Vector. As a result, a recombinant plasmid is created that expresses your siRNA in the desired host system. The siRNA cassette, once transferred, will become linked to the specific expression elements for which the Acceptor Vector was designed. For a complete list of Acceptor Vectors, visit our web site at localhost/clontech.

This vector also contains an ampicillin resistance gene, which is the marker for propagation and selection of the Donor Vector in E. coli. In addition, pSIREN-DNR contains the sucrase gene from B. subtillis (SacB), which provides negative selection against incorrect recombinants and the parental Donor Vector following recombination.

Use

RNAi-Ready pSIREN-DNR is used for targeted gene silencing when an oligonuceotide encoding an appropriate siRNA is inserted. To construct recombinant pSIREN-DNR, first design, generate, and anneal complementary siRNA oligonucleotides using the protocols in the BD? Knockout RNAi Systems User Manual (PT3739-1). The annealed oligonucleotide should contain 5'-BamH I and 3'-EcoR I sites. Then ligate the annealed oligonucleotide into RNAi-Ready pSIREN-DNR (provided in the BD Knockout Adenoviral RNAi System 2).

The resulting siRNA cassette can be transferred from pSIREN-DNR to pLP-Adeno-X-PRLS Viral DNA (or any Acceptor Vector) by Cre-mediated recombination as described in the BD Adeno-X Expression Systems 2 User Manual (PT3674-1). Recombinant clones can be selected by growth on media containing chloramphenicol and sucrose. After selection, recombinant plasmids can be further propagated in chloramphenicol or the antibiotic that is appropriate for the resistance marker of the Acceptor Vector.

To introduce the adenoviral siRNA construct into cells, refer to the BD Adeno-X? Expression System User Manual (PT3414-1).

Location of Features

• loxP recombination sites: 4834–4867; 902–935
• Chloramphenicol (Cmr) open reading frame (ORF): 873–214
• SacB negative selection marker: 944–2854
• Ampicillin-resistance gene:
• Start codon: 2990–2992; stop codon: 3848–3850
• pUC origin of replication: 3998–4641
• T7 RNA polymerase promoter: 4752–4770

• Human U6 promoter (PU6): 4877–5133

Primer Locations

• M13-reverse sequencing primer site: 65–50
  • 5'-AACAGCTATGACCATG-3'
• M13-forward sequencing primer site: 4726–4742
  • 5'-GTAAAACGACGGCCAGT-3'