Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 220

published literature [ 47 ]. In addition to the capsid mutation, it
may be desirable to introduce a second point mutation creating a
new restriction site not present elsewhere in the plasmid. This will
subsequently allow rapid screening of the presence of successful
mutagenesis by restriction enzyme digestion, rather than the need
for sequencing all clones.
Primers should have the following properties:

1. Be 25-45 bases long.
2. The desired mutation should be in the center of the primer.
3. The melting temperature should be greater than 78 °C (a
melting temperature calculator is available online at www.
genomics.agilent.com ) .
4. There should be a minimum GC content of 40 %.
5. The primer should end in one or more C or G bases.

Synthesis of the mutant DNA strand is generated by thermal
cycling, in which the plasmid is denatured; the primers containing
the mutation to be created anneal and are extended using PfuUltra
DNA polymerase. The QuikChange II XL kit provides complete
details of the reaction setup. The cycling parameters depend on the
plasmid size. General parameters are 95 °C for 1 min and then 18
cycles of 95 °C for 50 s, 60 °C for 50 s, and 68 °C for 1 min per
kb of plasmid length. End with a fi nal 7 min at 68 °C.

4.8.2 Thermal Cycling

After thermal cycling and placing on ice for 2 min, any remaining
original plasmid is removed by adding a Dpn I enzyme to digest
methylated DNA. As the template plasmid was generated in bacte-
rial cells, it will be methylated, while the new synthesized plasmid
containing the desired mutation will not be. The original plasmid
can therefore be targeted for removal by Dpn I. Add 1

4.8.3 Dpn1 Digestion

l of Dpn I
restriction enzyme to each reaction, mix thoroughly, and incubate
at 37 °C for 1 h.
2

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l of the Dpn I -treated DNA can subsequently be used for
the transformation of XL10-Gold cells as described in Sect. 4.1 .
Once clones have been cultured and plasmid isolated, if a restric-
tion site was also inserted in the mutagenesis, these clones can be
screened for the presence of the desired mutation using restriction
digestion prior to sequencing. Once presence of the mutation has
been confi rmed by sequencing, the plasmid can be used for viral
preparation as detailed above.

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5 Testing AAV Vectors In Vitro

Transducing cell lines with AAV vectors is an easy and relatively
quick method of assessing the effi cacy of vectors produced in the
lab. Since the aim of AAV retinal gene therapy may be to target a

5.1 Cell Lines