Use of pIRES Vectors to Express EGFP and Connexin Constructs in Studies of the Role of Gap Junctional Communication in the Early Development of the Chick Retina and Brain

Abstract

Control of cell proliferation is vital for the normal development of the neural retina. Gap junctional communication has been implicated in the control of retinal cell proliferation. We have previously shown that the expression of the gap junction protein Connexin 43 closely correlates with the first wave of cell proliferation in the retina. Preventing its expression using antisense oligonucleotides in the developing eye and surrounding tissues, produces a reduction in cell number and the formation of a small eye. In order to examine this in more detail we have developed a new means of manipulating connexin expression in the developing chick embryo. We have generated pIRES vectors which use cyclomegalovirus (CMV) to promote the expression of a green fluorescent protein (EGFP) and either wild type Cx43 or a dominant negative form of this connexin. Following injection of these constructs into the ventricles of the stage 10-11 chick embryo they can be incorporated into one side of the chick brain or optic vesicle using an electroporation technique, leaving the other side as a control. EGFP expression can be seen on the electroporated side of the chick brain within 24hours. Expression of the dominant negative construct in cultures of chick limb bud mesenchyme results in total block of cascade blue transfer when injected into transfected cells. Expression of both wild type and dominant negative constructs in the developing chick retina perturbs the normal development of the eye.

Key Words:

• Connexin
• development
• electroporation
• EGFP
• retina