Fig. 7. Determination of the connexin types expressed by differentiating neuroblasts by 7 DIV. A, Composite of gels from first-generation cDNA obtained by RT-PCR using universal primers, run on a 2% agarose gel, and visualized by ethidium bromide staining. Lanes 1, 3, and 5 show first-strand DNA from untreated cells, IL-7 alone, and IL-7 plus growth factors, respectively. Lanes 2 (untreated cells),4 (IL-7), and 6 (IL-7 plus growth factors) are negative controls, run in PCR without reverse transcriptase to verify that genomic DNA was not present. Lane 7 is the PCR product of Cx33 from mouse testis, and lane 8 is its corresponding negative control. Lane 9is the PCR product of Cx43 from rat heart, and lane 10is its corresponding negative control. B, Presence of Cx33 and Cx43 confirmed by RT-PCR. Second-generation PCR product was obtained with universal primers. Lanes 1, 3, and5 show the presence of restriction products of Cx43 afterHincII was used. The product gave two bands for untreated, IL-7 alone, and IL-7 plus growth factors. Lane 9 is the HincII digest of the PCR product from the rat heart, which also gave the characteristic double band for Cx43.C, Lanes 1, 3, and 5 show the presence of the restriction product of Cx33 afterMseI was used. This enzyme gave two bands for IL-7 alone and IL-7 plus growth factors. Lane 7 is the PCR product of the Cx33 from mouse testis that had been cut withMseI; two bands were formed. Sequencing the RT-PCR products revealed the presence of mouse Cx43 (99.1% identical) and Cx33 (98.2% identity with rat Cx33 sequence). Similar procedures applied with specific primers for Cx37 (D, lanes 1, 2) and for Cx40 (E, lanes 1, 2) in cells treated with IL-7 alone resulted in detection of a product of a 311 bp, consistent with the expression of Cx40. M, Molecular markers, PCR products of Cx37 (mouse brain; D, lane 1) and Cx40 (mouse heart; E, lane 1), respectively.