Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 214

4.1 Cloning
and Transformation

A typical example of a plasmid containing a GFP transgene between
AAV ITRs is shown in Fig. 3 . The recombinant expression cassette
of choice should be inserted into the plasmid using appropriate
restriction sites and ligated using DNA ligase. If the same restric-
tion sites are required at each end of the transgene insert, then the
digested plasmid backbone should be treated with alkaline phos-
phatase prior to ligation with the insert.
In its native form, the 145 bp AAV ITR forms a stable second-
ary structure (Fig. 4 ) and is therefore susceptible to deletion in the
cloning process. For this reason, we recommend the use of XL10-
Gold Ultracompetent bacteria (Agilent Technologies) for cloning
procedures, which we have found to be reliable at preserving ITR
integrity.

Fig. 3 Cloning map. Example of a plasmid containing AAV inverted terminal repeats with a green fl uorescent
protein coding sequence. Xma I restriction sites are annotated

Fig. 4 Inverted terminal repeat (ITR). Secondary structure formed by palindromic sequence of ITR. Image gen-
erated using Geneious software: Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M,
Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2012) Geneious v5.6,
available from http://www.geneious.com