Description of the method

The Transpose-Ad™ Adenoviral Vector System is an E. coli based system taking advantage of the site-specific transposition of the Tn7 transposon. The gene of interest is cloned into the MCS of the transfer vector, where it is flanked by two Tn7 inverted repeats (IRs). This creates a mini Tn7 transposable element. This construct is then transformed into chemically competent E. coli that contain two additional plasmids. The first is a helper plasmid expressing the Tn7 transposase open reading frame and whose products catalyze Tn7 specific transposition. The second is the Transpose-Ad™ 294 plasmid that contains the adenovirus genome, along with the target sequence for transposition (attTn7). The attTn7 is located strategically in a lacZ gene so that any positive recombinants can be scored as -gal^- colonies on plates containing Bluo-gal and IPTG.

White colonies are then grown and the purified recombinant Transpose-Ad™ 294 plasmid is transformed into chemically competent E. coli cells (HighQ-1™) for further purification. The purified recombinant Transpose-Ad™ 294 plasmid is then linearized with Pac I before being transfected into human QBI-HEK 293A cells, where the recombinant adenovirus will be produced and amplified.

Advantages

• No plaque purification necessary
• Uses site-specific transposition, a highly efficient means of recombination
• Rapid and convenient blue/white color screening method, no restriction enzyme mapping necessary
• No electroporation required
• The adenoviral backbone is already in the cells where transposition takes place and so does not require any additional transformation

Cell lines and viral particles

HighQ-1 Transpose-Ad™ 294 E. coli Cells

HighQ-1 Transpose-Ad™ 294 chemically competent cells harbor the Transpose-Ad™ 294 plasmid (Cm^R, carrying the resistance gene to chloramphenicol) and a plasmid encoding a trans-acting transposase (Tc^R, carrying the resistance gene to tetracycline). The transposition of the mini Tn7 element containing the cloned gene of interest takes place in the HighQ-1 Transpose-Ad™ 294 E. coli cells.

HighQ-1™ E. coli Cells

The DNA extracted from white colonies is re-transformed into the HighQ-1™ chemically competent cells to amplify the recombined Transpose-Ad™ plasmid and to segregate it from the transfer vector and the helper plasmid containing the transposase reading frame.

QBI-HEK 293A Cells

The QBI-HEK 293A cell line is a permanent line of primary human embryonic kidney (HEK) cells transformed by sheared human Ad5 DNA. These cells contain the E1A and E1B Ad5 viral genes, which complement the deletion of this essential region in the recombinant adenovirus. We have selected a superior sub-clone of HEK 293 that adheres strongly to plastic dishes and performs extremely well in plaque assay and transfection experiments. All our adenoviral vector systems use QBI-HEK 293A cells for viral replication and amplification.

QBI-Infect Viral Particles

The QBI-Infect is a first generation adenovirus expressing the bacterial LacZ gene under the control of the immediate/early CMV promoter (Ad5.CMVLacZE1/E3). This virus can be used to perfect adenovirus culture techniques before producing recombinant virus. People inexperienced with virology techniques can use QBI-Infect to prepare control plaques. Once amplified, QBI-Infect Adenovirus can be used to test the infectivity of cell lines or tissues other than QBI-HEK 293A cells or commonly used eukaryotic cells. The success of DNA transfer can be easily monitored with a substrate for -galactosidase. The QBI-Infect Viral Particles are included in all of our adenoviral vector systems.

Tn7 Transposon Theory

Transposons are DNA-segments that move (transpose) from one position to another either site-specifically or randomly. The transposition event uses a self-encoded recombinase called transposase. Transposons (or transposable elements) are widespread in nature and virtually every organism that has been examined carries transposable elements. In humans, 35-50% of the genome consists of transposable elements, or relics thereof.

Unlike most other transposons, Tn7, which is a bacterial transposon, transposes at high frequency into a specific site called attTn7. This site-specific insertion into attTn7 discourages any potentially harmful random insertions into bacterial chromosomes.

However, Tn7 resembles most other transposable elements in that it inserts into many different target sites if the attTn7 site is not available.

Tn7 encodes 5 different proteins that have different roles to play in the transposition. The Tn7 proteins TnsA, TnsB and TnsC form the core machinery, TnsABC, used in Tn7 transposition. The transposase is formed by TnsA and TnsB, whereas TnsC regulates Tn7 activity. TnsC plays the role of coordinator between the transposase and TnsD and TnsE.

In presence of TnsD, the transposon inserts site specifically into the attachment site of Tn7. If TnsE is used, transposition does not follow a site specific pathway. TnsE directs insertions into conjugable plasmids.

It is the ability to interact with TnsAB and the target DNA bound by TnsD or TnsE that confers the regulatory activity to TnsC. But this is not the only role played by TnsC. TnsC is also responsible for Tn7 immunity. This means that the presence of a copy of Tn7 in a DNA molecule does not allow a second Tn7 to transpose to this DNA molecule.

The Transpose-Ad adenoviral vector system takes fully advantage of the site-specificity of transposon Tn7. Since Tn7 has never been observed to insert into any site other than its attTn7 attachment site, it is not necessary to perform a lot of screening tests. Potential positive recombinants are b-gal- and are visualized as white colonies in presence of Bluo-gal and IPTG. And so far, all the white colonies picked that were resistant to Cm and sensitive to Amp and Tc have proven to be positive recombinants, thus almost completely eliminating the need for time consuming plaque purification.

References

Richards CA, Brown CE, Cogswell JP and Weiner MP. 2000. The Admid system: Generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli. BioTechniques 29:146-154.
The following are some major publications on Tn7.

General References to the Tn7 Transposon

Arciszewska, LK, D Drake, NL Craig. Transposon Tn7. cis-Acting sequences in transposition and transposition immunity. J Mol Biol. 1989. 207(1):35-52.

Bainton, RJ, KM Kubo, J Feng, NL Craig. Tn7 transposition: target DNA recognition is mediated by multiple Tn7-encoded proteins in a purified in vitro system. Cell. 1993. 72(6):931-43.

Barry GF. Abroad-host-range shuttle system for gene insertion into the chromosome of Gram-negative bacteria. Gene. 1988. 71:75-84.

Cleaver, SH, E Wickstrom. Transposon Tn7 gene insertion into an evolutionarily conserved human homolog of Escherichia coli attTn7. Gene. 2000. 254(1-2):37-44.

Craig, NL. Transposition. In Escherichia and Salmonella typhimurium: Cellular and Molecular Biology. Neidhardt, FC, Ingraham, JL, Low, KB, Magasanik, B, Schaechter, M, and Umbarger, HB (eds). Washington,D.C.: American Society for Microbiology, pp. 1996. 2339-2362.

Craig, NL. Target site selection in transposition. Annu Rev Biochem. 1997. 66:437-74. Review.
DeBoy, RT, NL Craig.Target site selection by Tn7: attTn7 transcription and target activity. J Bacteriol. 2000. 182(11):3310-3.

Flores C, MI Qadri, C Lichtenstein. DNA sequence analysis of five genes; tnsA, B, C, D and E, required for Tn7 transposition. Nucleic Acids Res. 1990. 18(4):901-11.

Flores CC, S Cotterill, CP Lichtenstein. Overproduction of four functionally active proteins, TnsA, B, C, and D, required for Tn7 transposition to its attachment site, attTn7. Plasmid. 1992. 28(1):80-5.

Gringauz, E, KA Orle, CS Waddell, NL Craig. Recognition of Escherichia coli attTn7 by transposon Tn7: lack of specific sequence requirements at the point of Tn7 insertion. J Bacteriol. 1988. 170(6):2832-40.

Kubo, KM, CNL Craig. Bacterial transposon Tn7 utilizes two different classes of target sites. J Bacteriol. 1990. 172(5):2774-8.

Peters, J.E. and Craig, N.L. Tn7: smarter than we thought. Nat Rev Mol Cell Biol. 2001 Nov;2(11):806-14. Review.

Richards, C A, Ch E, Brown, JP Cogswell, MP Weiner. The admid system: generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli. Biotechniques. 2000 29(1):146-54.

Waddell,CS , NL Craig. Tn7 transposition: two transposition pathways directed by five Tn7-encoded genes. Genes Dev. 1988. 2(2):137-49.

Ordering Information

The basic kit contains the following components:
pCR259 transfer vector
pCR276 transfer vector
HighQ-1 Transpose-Ad™ 294 cells
HighQ-1™ cells
QBI-Infect viral particles
Transfection reagents (2M CaCl₂, TE 0.1X and 2X HBS).

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