FU Berlin Digitale Dissertation
Jan Rathenberg : Gene targeting and single-cell electroporation to analyze cholinergic neurons using choline acetyltransferase as marker Gezielte Genmodifikation und Single-Cell Elektroporation zur Analyse cholinerger Neuronen mittels Cholin Acetyltransferase als Marker
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Abstract Cholinergic neurons use the neurotransmitter acetylcholine (ACh) and are involved in basic brain functions like learning and memory formation, awareness, the sleep and awaking cycle as well as motor behavior. Cholinergic neuron loss occurs during severe neurodegenerative diseases like Alzheimer or amyotrophic lateral sklerosis. So far immunohistochemistry or in situ hybridization against the cholinergic marker enzyme choline acetyltransferase (ChAT) serve to identify cholinergic neurons. However, these methods require fixation of the tissue and thus preclude in vivo experiments. One aim of the present work was to produce a fusion protein between ChAT and the green fluorescent protein (GFP) which will enable to visualize the cholinergic marker without fixation. Therefore the cDNA of ChAT was isolated from mouse spinal cord and subsequently the cDNA of GFP was cloned into a unique HindIII restriction site corresponding to the N-terminal part of the ChAT protein. The ChAT-GFP protein was expressed in COS-1 cells and primary cultures of hippocampal neurons as well as in hippocampal neurons of organotypic brain slice cultures. It was shown that the enzyme activity of the recombinant ChAT-GFP fusion protein is slightly reduced compared to the wildtype enzyme. However, it is still functional and similar distributed as the wildtype protein in cultured cells. In hippocampal neurons ChAT-GFP translocates into axonal processes and it is present in the presynaptic compartments. Since it was shown that ChAT-GFP is functional like the wildtype enzyme the cDNA of GFP was targeted to the ChAT gene of mouse embryonic stem cells by homologous recombination. These ChAT(GFP/+) ES cells were injected into blastozysts to produce chimeric mice. This blastozyst transfer provided 14 chimeric animals. Hetero- and homozygous mice which emerge from ChAT(GFP/+) ES cells will express the ChAT-GFP fusion protein in all cholinergic cells and hence these cholinergic neurons should show endogenous fluorescence. In parallel another ES cell clone was produced in which the first coding exon of ChAT is flanked by loxP sites (genotype ChAT(loxP/+)). This "knock-out" model will allow the conditional deletion of the ChAT gene specifically in the cholinergic nervous system during later developmental stages. A further aim was to establish organotypic brain slice cultures in order to study the physiology, development and degeneration of cholinergic neurons. With cultures prepared from ChAT-GFP targeted mice it will be possible to transfect cholinergic neurons. To this end a new method for electroporation of individual neurons, namely single-cell electroporation (SCE), was established and the efficiency of SCE was significantly improved. Using SCE, neuronal markers were expressed as GFP-fusion proteins in pyramidal neurons of organotypic hippocampal brain slice cultures and their distribution was compared with that of ChAT-GFP. It was shown that ChAT-GFP is present in the cytosol of the soma, dendritic spines and the axon but is absent from the nucleus and is at least in part associated with synaptic vesicles. The combination of gene targeting by homologous recombination and the direct gene transfer by SCE opens new avenues to analyze signal transduction in the cholinergic system at a molecular level.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title 0TITLE and APPENDIX 1INTRODUCTION 1.1 Organization of the cholinergic gene locus, mRNA transcription and translation 1.2 ChAT protein purification and characterization 1.3 The cholinergic synapse 1.4 The central and peripheral cholinergic nervous system 1.4.1 Anatomy 1.4.2 Development 1.4.3 Physiology 1.4.4 Disease 1.5 Organotypic cultures of neural tissue 1.6 Transfection of neurons by electroporation 1.7 In vivo imaging of the mammalian nervous system using fluorescent proteins 1.8 Cre/loxP mediated gene deletion or conditional gene "knock-out" 1.9 Creation of mutant mouse lines by homologous recombination 1.10 Aims of the project 2MATERIALS AND METHODS 2.1 Materials 2.1.1 Chemicals, reagents and consumable materials 2.1.2 Plasmid vectors 2.1.3 Bacteria strains, cell lines and animals 2.1.4 Primers and Oligonucleotides 2.1.5 Buffers and solutions 2.1.6 Media and solutions for bacteria, cell and tissue culture 2.2 Methods 2.2.1 Molecular Biology 2.2.1.1 Preparation of plasmid DNA 2.2.1.2 Determination of nucleic acid concentration 2.2.1.3 Sequence analysis 2.2.1.4 Digestion with restriction enzymes 2.2.1.5 Agarose gel electrophoresis 2.2.1.6 DNA fragment isolation 2.2.1.7 5´-Dephosphorylation 2.2.1.8 Ligation 2.2.1.9 Transformation 2.2.1.10 Preparation of competent cells 2.2.1.11 RNA preparation from mouse (C57Bl/6) spinal cord 2.2.1.12 Reverse transcription 2.2.1.13 Site-directed mutagenesis 2.2.1.14 PCR amplification of DNA fragments 2.2.1.15 PCR screen of ES cell clones in the 96 well format 2.2.1.16 Preparation of genomic ES cell DNA from 96 well culture plates 2.2.1.17 Southern transfer of DNA fragments onto membranes 2.2.1.18 Detection of PCR products of using the ECL system 2.2.1.19 Detection of genomic digestion fragments with radiolabeled 2.2.2 Biochemistry 2.2.2.1 Choline acetyltransferase activity assay 2.2.2.2 Determination of protein concentration 2.2.2.3 Protein gel electrophoresis 2.2.2.4 Staining of proteins in polyacrylamide gels 2.2.2.5 Western blotting and immunodetection 2.2.2.6 Fixation of cells and tissues 2.2.2.7 ChAT immuncytochemistry of transfected COS-1 cells 2.2.3 Cell culture 2.2.3.1 Transfection of COS-1 cells 2.2.3.2 ES cell culture 2.2.3.2.1 Preparation of feeder cells 2.2.3.2.2 Electroporation of ES cells 2.2.3.2.3 Picking of G418 resistant clones 2.2.3.3 Organotypic tissue culture 2.2.4 Electroporation of single neurons in brain slice cultures (SCE) 3RESULTS 3.1 Generation of expression and targeting vectors 3.1.1 ChAT-GFP and wildtype ChAT expression vectors 3.1.2 ChAT-GFP targeting vector 3.1.3 ChAT-loxP targeting vector 3.2 Functional characterization of the ChAT-GFP fusion protein 3.2.1 Expression of ChAT-GFP and wildtype ChAT 3.2.2 Enzyme activity of ChAT-GFP compared to the wildtype 3.2.3 Subcellular distribution of ChAT-GFP and wildtype ChAT in COS-1 cells 3.2.4 Subcellular distribution of ChAT-GFP in hippocampal neurons 3.3 Transfection of individual neurons in organotypic hippocampal slice cultures by single-cell electroporation (SCE) 3.4 Expression of presynaptic and postsynaptic marker proteins 3.5 Generation of ChAT-GFP and ChAT-loxP targeted mouse E14 stem cell 4DISCUSSION 4.1 A recombinant ChAT-GFP fusion protein has been generated which is efficiently expressed in COS-1 cells 4.2 Influence of GFP insertion in ChAT enzyme activity 4.3 Distribution of wildtype ChAT and ChAT-GFP in COS-1 cells 4.4 ChAT-GFP is expressed in pyramidal neurons and appears to be associated to synaptic vesicles 4.5 Establishing organotypic brain slice cultures 4.6 Improved single-cell electroporation (SCE) is a powerful transfection method for single neurons 4.7 Gene transfer of fluorescent proteins via SCE 4.8 Modification of the ChAT gene in ES cells by homologous recombination 4.9 Outlook 5SUMMARY 6ZUSAMMENFASSUNG 7ABBREVIATIONS 8REFERENCES

More Information:
Online available:	http://www.diss.fu-berlin.de/2002/194/indexe.html
Language of PhDThesis:	english
Keywords:	ChAT, GFP, gene targeting, single-cell electroporation, neuron
DNB-Sachgruppe:	32 Biologie
Date of disputation:	13-Sep-2002
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Priv.-Doz. Dr. Veit Witzemann
Second Referee:	Prof. Dr. Ferdinand Hucho
Contact (Author):	jrathenberg@web.de
Contact (Advisor):	witzeman@mpimf-heidelberg.mpg.de
Date created:	19-Sep-2002
Date available:	23-Sep-2002

 

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