Of these NKCC1b is also known to be found in brain RNA (Gamba, 2005). It is noted here, though, that the two isoforms of the NKCC1 cotransporter is found only in the European eel (Anguilla anguilla) as per research of Cutler and Cramb, 2002. Nevertheless, there is ample evidence that NKCC1, in human and other mammalian species, is functionally implicated in CNS cells. It is observed by Gamba, 2005, that the NKCC1 cotransporter is activated by receptors and assists in neurotransmission by driving anions into the cell. It is also observed by Strange et al, 2000, that the work of the NKCC1 cotransporter complements that of the KCC2 one. The choice of the culture medium, the neuronal-specific CAD cell line, and the somewhat CNS-specific NKCC1 dovetails perfectly for a research attempt that seeks to establish new facts on the molecular identity and other expression patterns of these unique electroneutral cotransporters in cells of the central nervous system (CNS). G. Gamba's excellent 2005 review article on these cotransporters has been extensively used in this paper because it is the most comprehensive document prepared to date being inclusive of all aspects described so far.
RT-PCR analysis was carried out...
ers has been extensively used in this paper because it is the most comprehensive document prepared to date being inclusive of all aspects described so far.
RT-PCR analysis was carried out on the murine neuronal cell line CAD on both differentiated and undifferentiated cells. Isolated RNA from both differentiated and undifferentiated cells was used. Annealing temperatures used were at C - C at 40 cycles for a maximum of one minute. Primers 10bp in length were used with a gc content above 50%, as recommended (Ribicki, 2001). Upon gel electrophoresis of all the products it was revealed that a 286 bp bit, exactly as long as the PCR insert, was apparent in all the gels for both differentiated and undifferentiated cells at all PCR temperatures. The insert was perfectly complemented once, as planned, and the amplicon was evident in clear form. There was no other unplanned product. It is thus concluded that the CAD cells, differentiated and undifferentiated, show definite expression of the NKCC1 cotransporter and that the SLC12A2 gene is present in activated form in both differentiated and undifferentiated CAD cells. There was no difference in levels of expression in both types of cells. Since there is no previous report of this cotransporter being expressed in this particular cell line there is scope for using these cells in future to study NKCC1 cotransporter expression. This is also interesting that the cotransporter, being somewhat neuronal specific, was expressed in both types of cells though it is well-known that the undifferentiated CAD cells lack many truly neuronal structures and properties evident in the differentiated cells. Thus, the implications of the cotransporter being expressed in both types of cells mean that both types can be used in future