The exogenous expression of each lens connexin in lens primary cultures was attempted by using our previously developed retroviral approach.
25 High-titer retroviruses—RCAS(A), RCAS(A)-Cx43, RCAS(A)-Cx45.6, and RCAS(A)-Cx56—were used to infect monolayer primary lens cultures. Seven days after the infection, the expression of the three exogenous connexins was clearly demonstrated by Western blot analysis
(Fig. 1) and immunofluorescence studies
(Fig. 2) . Exogenous Cx43 (
Fig. 1 , lane 1), Cx45.6 (
Fig. 1 , lane 2), and Cx56 (
Fig. 1 , lane 3) were abundantly expressed in lens primary cultures, as detected by anti-epitope tag FLAG antibody. No band was detected from cultures infected with retrovirus, RCAS(A) (
Fig. 1 , lane 4). Exogenous connexins were expressed in multiple migrating forms, as detected by SDS-PAGE. Similar migrating patterns have been shown for their endogenous counterparts, expressed in primary lens cultures, as a result of posttranslational phosphorylation.
34 35 Immunofluorescence experiments using anti-FLAG epitope tag antibody revealed the localization of exogenously expressed lens connexins
(Fig. 2) . All three exogenous connexins were expressed in lens epithelial cells, which were revealed by immunofluorescence signals as punctuate spots surrounding the cells
(Figs. 2a 2c 2e) , which is the typical expression pattern of gap junctions. Exogenous Cx45.6 and Cx56 were also expressed in abundance within lentoid bodies
(Figs. 2d 2f) . Cx43, in comparison with Cx45.6 and Cx56, was expressed to a lesser extent in the lentoid bodies, and the expression was mostly localized close to the edges and surface areas of the lentoids
(Fig. 2b) . After 3 days of infection, more than 95% of the cells on the culture plate contained exogenously expressed connexins.