Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer

The regenerating amphibian limb provides a useful system for studying genes involved in the establishment of positional information. While a number of candidate genes that may playa role in pattern formation have been identified, their function in vivo is unknown in this system. To better ascertain the role of these genes, it would be useful to be able to alter their normal patterns of expression in vivo and to assess the effects of this misexpression on limb pattern. In order to achieve this, a method of introducing a plasmid containing the eDNA of a gene of interest into a newt blastema (a growth zone of mesenchymal progenitor cells) is needed. Unfortunately, most commonly used transfection techniques cannot be used with newt blastema cells. In this study, I have used the techniques of lipofection and direct gene transfer to introduce plasmid DNA containing reporter genes into the cells of a regenerating newt limb. The technique of lipofection was most effective when the blastema cells were transfected in vitro. The optimal ratio for transfection was shown to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA present in the mixture (1:3 ratio maintained) resulted in a corresponding increase in gene expression. The technique of direct gene transfer was used to transfect newt blastema cells with and without prior complex formation with Lipofectin. Injection of plasmid DNA alone provided the most 3 promising results. It was possible to introduce plasmid DNA containing the reporter gene ~-galactosidase and achieve significant gene expression in cells associated with the injection site. In the future, it would be interesting to use this technique to inject plasmid DNA containing a gene which may have a role in pattern formation into specific areas of the newt blastema and to analyze the resulting limb pattern that emerges.

Perturbing the impulse activity of a single identified neuron augments the formation of long-term memory in a molluscan semi-intact preparation /

The aim of this study was to investigate the neural correlates of operant conditioning in a semi-intact preparation of the pond snail, Lymnaea stagnalis. Lymnaea learns, via operant conditioning, to reduce its aerial respiratory behaviour in response to an aversive tactile stimulus to its open pneumostome. This thesis demonstrates the successful conditioning of na'ive semiintact preparations to show learning in the dish. Furthermore, these conditioned preparations show long-term memory that persists for at least 18 hours. As the neurons that generate this behaviour have been previously identified I can, for the first time, monitor neural activity during both learning and long-term memory consolidation in the same preparation. In particular, I record from the respiratory neuron Right Pedal Dorsal 1 (RPeD 1) which is part of the respiratory central pattern generator. In this study, I demonstrate that preventing RPeDl impulse activity between training sessions reduces the number of sessions needed to produce long-term memory in the present semi-intact preparation.

Trilobites of the Upper Cambrian (Marjuman) Pika formation of the Southern Canadian Rocky Mountains

The Upper Cambrian Pika Formation in the southern Canadian Rocky Mountains forms a complete lithologic Grand Cycle. The overall pattern of deposition is one of shallowing upwards from a subtidal, muddy, storm-influenced basin to a shallow carbonate bank. The Pika passes gradationally into the overlying inter- to supratidal siliciclastics of the Arctomys Formation. This transition probably reflects a fall in relative sea level. 2 Twenty seven collections from three sections yielded trilobites. The faunas are assigned to two low-diversity biofacies: the Marjumia - Spencella Biofacies and the GZyphaspis - menomoniid Biofacies. In contrast to biofacies of deeper, open-shelf environments, such as the Wheeler and Marjum formations of Utah, the Pika biofacies lack agnostid trilobites. Consequently, agnostid-based zonations defined elsewhere in North America cannot be applied to the Pika and a new sequence of three zones and one informal fauna is proposed for use in inner shelf facies. Eleven species belonging to six genera are described and illustrated. The species Marjumia bagginsi is new. Other genera present are: Bolaspidella, Knechtelia, GZyphaspis and Spencella, in addition to a number of indeterminate forms