Restriction endonuclease pme 1

Recombinant Ad-GluR6c-green fluorescent protein constructs were produced in accordance with standard techniques (He et al., 1998). The pAd Track CMV vector is bicistronic, and expresses both green fluorescent protein and the GluR6c domain. Briefly, GluR6c (852-908 amino acids of GluR6) was generated by polymerase chain reaction of the appropriate GluR6c coding region to incorporate lanking Bgl II and Hind III sites followed by ligation into the Ad shuttle vector pAdTrack-CMV digested with Bgl II and Hind III (Promega). The resultant plasmid was linearized by digestion with restriction endonuclease Pme I (New England Biolabs, Beverly, MA), and subsequently cotransformed into Escherichia coli (Promega). BJ5183 cells (Addgene, Cambridge, MA, USA) have an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected with kanamycin, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into Ad packaging cell lines, Human Embryonic Kidney 293 cells (Addgene). Recombinant Ads were generated typically within 7 to 12 days, purified, and then tittered.