Adenoviral Vector System
The Transpose-Ad Adenoviral Vector System is a Tn7-based transposition system for generating recombinant Adenoviruses in E. coli (1). In this novel system, a complete expression cassette containing a gene of interest is first introduced via cloning steps in a mini transposon (mini Tn7) located in an Adenovirus transfer vector (figure 1). The target site for Tn7 insertion, attTn7, is located on the Transpose-Ad 294 plasmid, which also contains the E1-E3 Adenovirus serotype 5 genome. To simplify the process of generating a recombinant adenovirus, the Transpose-Ad 294 plasmid is carried within HighQ-1 Transpose-Ad 294 competent cells thus eliminating a co-transformation step.
Transposition from the Adenovirus transfer vector to the Transpose-Ad 294 plasmid occurs following the introduction of the transfer vector into HighQ-1 Transpose-Ad 294 bacteria. In addition to the Transpose- Ad 294 plasmid, this bacterial host strain carries a plasmid encoding a trans-acting Tn7 transposase. The transposition-specific attachment site is strategically positioned in the lacZ gene (lacZattTn7) cloned in the deleted E1 region. Transposition into lacZattTn7 disrupts the lacZ reading frame, thus allowing for the screening of recombinant Transpose-Ad plasmids containing the mini-Tn7 expression cassette in a single step that is efficiently and easily scored as a ß-gal- (white) phenotype on plates containing Bluo-gal and IPTG.
DNA extracted from white colonies is re-transformed into chemically competent HighQ-1 cells in order to segregate and amplify the recombinant Transpose-Ad plasmid from the Adenovirus transfer vector and the plasmid containing the transposase. The segregated recombinant Transpose-Ad plasmid is then purified in large quantities, linearized with Pac I and transfected into QBI-HEK 293 cells to generate a recombinant Adenovirus expressing the desired gene product.
pCR276 Transfer Vector: designed to facilitate expression from a user-supplied promoter. It contains a multiple cloning site and the SV40 poly A signal. The user-supplied promoter and transgene are cloned into the multiple cloning site in front of the poly A sequence.
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