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This thesis describes new aspects in the development of the in-utero electroporation method (IUE) and hereby refers to the neurogenesis and the cortical migration in the central nervous system. With the assistance of the described transfection technique the development of neuronal stem cells could be manipulated in iso- and allocortex by regulating the expression of defined plasmides.
The first section of this thesis examines the functionality and the validity of the IUE in the C57BL/6 wildtype mouse in different embryonic stages between E12 and E17. Next were defines standard values for electroporation and transfection. In the process, various plasmids were tested in vitro and in vivo and necessary voltages and injection points examined for a successful transfection. The success could be measured with fluorescent signals and the number of transfected cells.
The operative parameters with the least lethality but a high effectiveness of the transfections of embryos were determined for the execution in the in−utero electroporation. An atlas for transfection of various brain regions with the corresponding electrode angles was developed in the C57BL/6 mouse model.
Transfections with 2 different plasmids in the same embryo were also executed successfully.
This showed that the in-utero electroporation in different hemispheres can be ideally utilised to compare the effects of the transfected plasmids. However, the transfection of two different plasmids in the same hemisphere represents an optimal analysis of the interaction of the encoded proteins.
In order to analyse cortical migration, histogram evaluations after in-utero electroporation was used for the first time in this project. For this purpose, somatosensory cortex regions of the cerebrum were manipulated in various embryonic stages and the results in the distribution of the fluorescent signals and the transfected cell bodies compared. It became evident that the distribution of the manipulated cell bodies captured by the count provided the same or similar results in the last quarter of gestation with histogram calculations of the fluorescence in the tissue section at transfections. This new analysis procedure of the neuronal migration through histogram analyses was successfully tested in this dissertation on two neuG ronal cell membrane proteins, namely the PRG-1 and the LPP-1 with splicing variation LPP-1a.
PRG-1 was discovered as participating membrane protein some years ago during the research
of molecular impacts in the regulation of the axonal outgrow in the hippocampus formation.
For the first time, this dissertation examined the migration of neuronal post-mitotic cells in the somatosensory cortex region of the cerebrum, influencing the membrane protein PRG-1 in the embryonic phase. Overexpression of PRG-1 in progenitor cells via in-uteroelectroporation reduced the number of postmitotic cells in the cortical plate. The majority of the transfected cells, that over-expressed PRG-1, could be found in the ventricular and subventricular zone. Only few transfected post-mitotic neurones were between the subventricular and intermediary zone in the transition phase at a cellular deficiency of PRG-1, which represents the beginning of the radial migration. Even though PRG-1 does not possess any known enzymatic activity, there were indications that PRG-1 could have a regulatory function in the migration of post-mitotic neurones.
The impact of neurogenesis and neuronal migration, caused by the transfection of LPP-1 and LPP-1a encoded plasmids, was examined in the last section of this dissertation. Both proteins dephosphorylate active lipid phosphates and are important for the development of the cerebral cortex with the functional maturing of the neurones. It was determined that down- as well as a upregulation of these proteins only resulted in an almost similar distribution of the transfected cells when compared to the control. This could be an indication that the balance of the metabolic product concentration (LPA, S-1-P, C-1-P and LP) of LPP-1 and LPP-1a is of significance for the neuronal migration of post-mitotic neurones in the somatosensory cortex in the micro-milieu of the surrounding cells.