Transgene Labelling In Specific Neurons Of C.elegans With Different Methods

The C.elegans has been widely used as a modle organism by researchers in biologicalscience. There are sevaral resons for its widespread use: the simple body structure, shortlife circle, high breed compentency and the whole genome sequenced already. Comparedwith other modle organisms, the neuron system of C.elegans is the simplest: there are only302neurons which can be devided into interneuron, sensory neuron and moter neuron andso on. The research is meant to make sure the exact position of the neurons by applyingthe neuron system of C.elegans, so that we can provide the basic and importantinformation for the neurons’ functions in the neural circuit. Very few technologies can beapplied to mark one single neuron, although the technologies for genes’ location in liveworms are quite numerous. The main reason may be that the genes’ expression inC.elegans is wide range and the promoters which can locate in particular neurons arelimited. Given that, both molecular biological methods (including the FLP/FRT system,Cre/LoxP system,2A system, R/RS system) and confocal laser scanning technique havebeen combined to confirm the promoters that promote fluorencent proteins’ expression incertain neurons, and then the digital worms based on the information of neurons’ positionwill be a reality.On the other hand, some wonderful tools and skills for constructing plasmids duringmarking the specific neurons have been found out, particularly the combination ofin-fusion method and multisite gateway technique, not only shows the high effectivenessand seamless ligation in in-fusion reaction, but also takes great advantages of repeated usein multisite gateway reaction. At the same time, by applying this method to genes’conditional expression, further study on the neurons’ and the genes’ function in thebehavior of C.elegans. can come true. Following locating neurons, other optogeneticproteins such as ChR2and Ca2+protein G-CaMP might express in these neurons in orderthat more functions of neurons can be studied further."Multi-gene expression under the control of one single promoter" is also known as"polycistronic expression strategy". So far, only the spliced leader (SL2) based ontrans-splicing has been adopted in C.elegans, but its use is a little limited by the disadvantage of irregular expression in worms due to the subtypes of SL2. Here a new"polycistronic expression strategy" is developed, that’s the expression vector based onself-cleaving2A peptide. This strategy can make different proteins express respectively ina single ORF while stop condon is absent. The efficiency of2A peptide has been tested inZebrafish, Mice and cultured cells. P2A (porcine teschovirus-12A) is applied in theexperiment to confirm whether it can come into use in C.elegans. The preliminary datashow proteins can express in C.elegans, so that a new method appears in constructingpolycistronic vectors.