Transpose-Ad™

Q. Is this technology described in a recent publication?

A. 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.

Q. Is the Transpose-Ad™ system the same as the Admid system?

A. Yes, Qbiogene has a license from GlaxoSmithKline to use and sell the technology. Qbiogene has developed the technology into a comprehensive and easy-to-use kit complete with ready-to-use, quality controlled components.

Q. I am just getting into adenovirus work and would like to know which kit I should start working with?

A. The Transpose-Ad™ is an excellent technology to familiarize yourself with Adenovirus. This system includes a convenient blue/white screening method that makes the identification of positive recombinant Adenovirus plasmids very easy, fast and reliable.

Q. What is the frequency of transposition in this kit?

A. It varies between 9 and 40%, averaging at 23% of white colonies.

Q. What is the advantage of using transposition versus homologous recombination?

A. The Tn7 transposon is particularly interesting because it can transpose at a high frequency to a specific target site. In E. coli this target site is called attTn7. This means that in the presence of attTn7 in E.coli, Tn7 insertion is site-specific and therefore a positive recombinant is pure (100%) and does not need to be purified by plaque assay.

Q. How much transfer vector (pCR259, pCR276) is included in the kit?

A. 25 µg of each.

Q. Why does one have to screen with Cm, Tc and Amp?

A. Because transformed HighQ-1 Transpose-Ad™ cells contain:

- the helper plasmid which confers resistance to tetracycline (Tc^R)
- the Transpose-Ad™ which confers resistance to chloramphenicol (Cm ^R)
- the recombinant shuttle vector which confers resistance to ampicillin (Amp ^R)

Recombinants are Tc^S, Cm^R, and Amp^S

Q. I was told transposons insert just about anywhere in the genome.

A. When attTn7 is unavailable, Tn7 does resemble most other transposable elements, inserting at low frequency into many different target sites. In the presence of attTn7, transposition is site-specific; the Tn7 inserts at high frequency into this specific site in E. coli. The Transpose-Ad™ 294 plasmid carried in the HighQ-1 Transpose-Ad™ 294 E. coli cells does contain the attTn7 target site. Tn7 has never been observed to insert in any other region in the presence of attTn7.

Q. What are the deletions in the Transpose-Ad™ 294 plasmid?

A. E1 deletion: 356-3533. E3 deletion: 28599-30469

Q. What is the size of the helper plasmid?

A. The helper plasmid is 13.2 kb.