4.1. Plasmid Construction

Complementary DNA (cDNA) for both sheep Cx46 (sCx46, also known as Cx44) and Cx50 (sCx50, also known as Cx49) were synthesized and each of them was inserted into an expression vector, pIRES2-EGFP, with an untagged GFP reporter between the restriction enzyme sites, XhoI and EcoRI (NorClone Biotech Laboratories, London, ON, Canada), as previously described [47,48]. The Cx46 and Cx50 variants (Cx46 L10I, N13E, A14V, Q15N and Cx50 I10L, E13N, V14A, N15Q, and S89T) were generated by PCR-mediated site-directed mutagenesis using the corresponding untagged wild-type constructs as templates with the appropriate primers. All wild-type and variant constructs were sequenced to confirm the accuracy of the nucleotide sequences. The primers used to generate these point variants are listed below:

Cx46 L10I1: forward 5′ GC TTC CTG GGG AGA ATC CTA GAG AAC GCC CAG 3′ and reverse 5′ CTG GGC GTT CTC TAG GAT TCT CCC CAG GAA GC 3′

Cx46 N13E: forward 5′ GG AGA CTC CTA GAG GAG GCC CAG GAG CAC TCC 3′ and reverse 5′ GGA GTG CTC CTG GGC CTC CTC TAG GAG TCT CC 3′

Cx46 A14V: forward 5′ CTC CTA GAG AAC GTG CAG GAG CAC TCC 3′ and reverse 5′ GGA GTG CTC CTG CAC GTT CTC TAG GAG 3′

Cx46 Q15N: forward 5′C CTA GAG AAC GCC AAT GAG CAC TCC ACT G 3′ and reverse 5′ C AGT GGA GTG CTC ATT GGC GTT CTC TAG G 3′

Cx50 I10L: forward 5′ GT TTC CTG GGG AAC CTC TTG GAG GAG GTG 3′ and reverse 5′ CAC CTC CTC CAA GAG GTT CCC CAG GAA AC 3′

Cx50 E13N: forward 5′ G GGG AAC ATC TTG GAG AAC GTG AAT GAG CAC 3′ and reverse 5′ GTG CTC ATT CAC GTT CTC CAA GAT GTT CCC C 3′

Cx50 V14A: forward 5′ C ATC TTG GAG GAG GCC AAT GAG CAC TCC ACG 3′ and reverse 5′ CGT GGA GTG CTC ATT GGC CTC CTC CAA GAT G 3′

Cx50 N15Q: forward 5′ C TTG GAG GAG GTG CAG GAG CAC TCC ACG 3′ and reverse 5′ CGT GGA GTG CTC CTG CAC CTC CTC CAA G 3′

Cx50 S89T: forward 5′ GTC TCC ACG CCG ACG CTG GTG TAC GTG 3′ and reverse 5′ CAC GTA CAC CAG CGT CGG CGT GGA GAC 3′