Write 35 pages with APA style on Molecular Identity of Co-transporter KCC in Differentiated and Undifferentiated Mouse Neuronal Cell Line CAD.

Write 35 pages with APA style on Molecular Identity of Co-transporter KCC in Differentiated and Undifferentiated Mouse Neuronal Cell Line CAD. The cotransporter KCC2 is evident in neuronal processes with a specific preference for inhibitory ones where magic action is relevant. KCC2 action precedes the ergic one as the KCC2 action brings down the intracellular chloride concentration to optimum levels to make it possible for ergic action. The phylogenetic, molecular and structural-functional details of the cotransporters have also been provided with special emphasis on KCC2. The CAD cell line, new in studies like this, has been introduced. The cell line exhibits catecholaminergic voltage-dependent currents across membranes. Specific experimentation details have not been provided. A 5708 nucleotide region of the SLC12A5 gene that encodes the KCC2 cotransporter protein was probed with two 20 bp bits, left and right primer details in Appendix, with RT-PCR analysis. The resulting 206-nucleotide insert was perfectly amplified with one compliment in the RT-PCR at temperatures varying from C - C. mRNA isolated from undifferentiated and differentiated murine CAD cells were used for the analysis. Electrophoresis of the RT-PCR products through 1.5% agarose gel revealed that most of the inserts had been perfectly complemented once during the RT-PCR producing evidence that the SLC12A5 gene is present in both murine differentiated and undifferentiated CAD cells and that these cells can, thus, later be used as culture media for further study of the KCC2 cotransporter expression and molecular identity. This is of great importance to such study as a suitable cell line has been hard to find so far and the easy manner in which the gene revealed itself in this study assures that this cell line can be a very convenient medium of further such studies. Seven electroneutral cation-coupled co-transporters have been found in the physiological systems of mammals to date (Delpire, E., 2000). These, together with two orphan members, belong to the SLC12 gene family (Gamba, G., 2005) and cloning of the cDNA encodes the different members. These are all sodium/potassium-coupled chloride cotransporters described succinctly below. They can be functionally classified broadly into four groups according to the cation coupling to the chloride ion, the stoichiometry of the transport process and the relative sensitivities to inhibitors.

Contents:

1. Introduction 8-9

1.1 CAD Cells 9-10

1.2 The Cotransporters (KCC1, 2, 3 & 4) 10-11

1.3 The Electroneutral Cotransporters 11-12

1.4 Cotransporters KCC1 11-15

1.4.1 Genetic Details

1.4.2 Structure

1.4.3 Function and Location

1.5 Cotransporter KCC3 15-16

1.5.1 Genetic Details

1.5.2 Location

1.6 Cotransporter KCC4 16-17

1.6.1 Molecular Genetics with Structural Identity

1.6.2 Function and Location

1.7 Cotransporters: General Features 17

1.8 Cotransporter KCC2 17-24

1.8.1 Phylogenetics

1.8.2 Structural and Locational Implications

1.8.3 Structural and Locational Implications

1.8.4 Specific Neuronal Location

1.8.5 Regulatory Action

1.8.6 Neuron-Specificity

1.8.7 Post- and PreNatal Concentration

1.8.8 Immunohistochemical Analysis

2. Methodology

2.1 RNA Isolation Technique 24

2.2 RNA Isolation Protocol 24-27

2.3 Estimation of RNA Yield 27-29

2.3.1 Vol. Isolated RNA in Differentiated Cells

2.3.2 Vol. Isolated RNA in Undifferentiated Cells

2.4 Primer Design 29

2.5 The PCR Analysis 29-30

2.6 Gel Electrophoresis Technique 30-31

3. Results 31-41

4. Discussion 42-43

5.