This paper introduces a research project that shall investigate the expression patterns of the NKCC (Sodium-Potassium-coupled Chloride) Cotransporters in a newly-developed murine neuronal cell line CAD (caspase-activated deoxyribonuclease) to try and establish molecular identity…
This property is of special significance to the project as the emphasis of research is on the NKCC1 member, one that is known to have two isoforms – NKCC1a and NKCC1b. 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 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 ...
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The new cell line has been introduced hereafter and it is noted here that, significantly, it is one of the few thoroughly neuronal cell lines that can be induced to reversibly differentiate under culture conditions. This property is of special significance to the project as the emphasis of research is on the NKCC1 member, one that is known to have two isoforms - NKCC1a and NKCC1b.
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