Figure 1. Generation of two transgenic mouse models overexpressing Cx43 mutants.

(A) The membrane topological structure of Cx43 was generated by Microsoft Office Visio. R76W mutant (amino acid residue arginine-76 (R) is replaced by tyrosine (W)) (indicated by solid black circle) is located in the interface of second transmembrane and first extracellular loop domain. Δ130-136 mutant (deletion of amino acid residues 130 to 136) is located in the cytoplasmic loop domain. N, NH₂-terminus; C, COOH-terminus; E1, first extracellular loop domain; E2, second extracellular loop domain; CL, cytoplasmic loop domain. (B) The DNA constructs used to generate two transgenic mouse models consist of 10-kb DMP1 promoter and exon 1 (E1), and followed by an intron, a full-length Cx43 dominant negative mutant and GFP. (C) Genome DNA was extracted from tails of WT and the two transgenic mice. A PCR product of 770 bp pointed to endogenous Cx43 in WT mice and an 1813 bp and 1833 bp to R76W and Δ130-136 mice, respectively (left panel). A size difference of 20 bp was detected between R76W and Δ130-136 mutants (right panel). (D) The gene copy numbers were determined using qPCR using genomic DNA isolated from tails of two homozygous transgenic mice. (E) Lysates of tibial bone from WT and the two transgenic mice were prepared and immunoblotted with anti-GFP or anti-β-actin antibody followed by corresponding HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody. (F) The primary osteocytes were isolated from WT and the two transgenic mice, and were microinjected with Alexa594 dye. Gap junction intercellular coupling was blocked in osteocytes from the two transgenic mouse models, but not from WT mice. The original injected cell was indicated by “*”. (G) The primary osteocytes were isolated from WT and the two transgenic mice, then were subjected to flow shear stress (FSS) for 15 min or non-FSS conditions. Dye-uptake assay was performed with ethidium bromide (EtBr). Hemichannel dye uptake was only blocked in Δ130-136 mice, but elevated in osteocytes from R76W mice compared to WT. Data shown are mean ± SD. **, P < 0.01; ***, P < 0.001.