AdEasy™

Q. Will extra bases in the 5' and 3' UTRs (Untranslated regions) of my gene cloned into a transfer vector affect protein expression?

A. For optimal protein expression, UTRs have to be as small as possible, especially in 5' to avoid formation of secondary structures in the mRNA. A minimal length (7-8 bases) in front of the ATG is acceptable, especially if the Kozak consensus sequence (GCCGCCACCATGG) is included. There are no special requirements for the 3'UTR.

Q. Is it possible to first transform the BJ5183 with pAdEasy-1 and then make this recombinant strain electrocompetent for transformation with the linearized transfer vector?

A. Yes, it is possible. However, we do not recommend it since BJ5183 cells support the RecA recombination system. Our researchers have shown the occurrence of unwanted recombinations in laboratory tests. Therefore, we recommend growth and amplification of plasmids should not be performed in that strain of bacteria. That is why the DH5 strain is also included in the AdenoVator™ kit is that it allows amplification without rearrangements (does not support recombination).

In the following reference, researchers have first introduced the viral plasmid into BJ5183 cells and then electroporated the recombined transfer vector into the cells:

Zeng, M. et al. (2001). AdEasy system made easier by selecting the viral backbone plasmid proceeding homologous recombination. BioTechniques 31:260-262.

Q. What is the best way to screen for recombinant DNA made with the AdEasy™ kit?

A. The restriction enzyme Bst XI gives a very good diagnostic pattern to screen recombinants with the AdEasy™ system. The restriction fragments of the pAdEasy-1 plasmid are as follows:

1- 11921 bp
2- 8237 bp*
3- 5251 bp
4- 4254 bp
5- 2379 bp
6- 1399 bp

Fragment number 2 is the only one that will vary with the size of insert and from one transfer vector to another. With pShuttle-CMV, it shifts up to about 11-12 kb (and higher with the insert) whereas with pShuttle, it shifts to approximately 10.5 kb. This band may sometimes be confused with band 1 but disappearance of band 2 is a good indication of positive recombinant. This, in conjunction with the Pac I restriction pattern, makes identification of positive recombinants very straightforward.

Q. Upon screening recombinants with Pac I, some clones give a 30-35 kb band and a 3.0 kb band, others the same 30-35 kb and a 4.5 kb band. Which one is the good pattern?

A. Some recombinants might come from a recombination event between the right side homologous regions and the origins of replication instead of with the left homologous regions (see applications manual). These 2 recombinants are equally good and can very well be used but will give different restriction patterns. In our own experience, the pattern giving a band at 4.5 kb is generally the most abundant.