The Establishment And Application Of C. Elegans Neuron-specific Expression System

Caenorhabditis elegans is an important model organism. The characteristics ofsimple structure, short life cycle, and strong reproduction，make it be utilized inneurobiological research. The simple nervous system can be classified into several groups:motor neurons, sensory neurons, intermediate neurons and so on. Generally, expression ormarking in specific-neuron for targeting research is shown important in C.elegans neuronresearch. However, in C. elegans, specifically marked neurons are few and there is ashortage of avaliable technologies. And the vector construction is labor-consuming andtime-costing. The basic starting point of the study is to solve these problems.In the study, C. elegans single neuron expression promoters were constructed to thefirst entry clones of Multisite Gateway system, so that expression clones can beconstructed efficiently and accurately. The problem encountered in this process is that theBP reaction for large DNA fragments was low efficiency but high cost. So, an intermediatecloning vector was constructed for”in-fusion” method carrying out to establishing a entryclone library of neuron-specific expression promoters. Consequently, these promoterswere wide versatility that can transform into expression vector by combining with anygene. To verify the exressed area in C. elegans, these promoters were connected withtetanus toxin gene (TeTx), function gene, or fluorescence protein gene. And theexpressions drived by promoters which have the same expression profile were compared.However, in C.elegans, neurons with specific marker whose expression drived by singlepromoter are limited. It is reported that they are less than20. For the purpose of markingneurons with multiple promoters, site-specific recombinase FLP/Frt and Cre/loxPsystems were indtroduced. By using the recombinase systems and modified destinationvector for Multisite Gateway technology in combination, neurons can be marked byoverlapping expression promoters. To achieve expressing in the same organization of C.elegans, the spliced leader2(SL2), a produnction of the second form of trans-splicing inworm, was applied. The effect of polycistronic expression mediated by SL2e whichfollowed by fluorencent protein was analysis by the method of laser scanning confocalimaging. The neuron-specific expression system established can be conveniently applied to theexperiments for studying the molecular mechanisms of C. elegans neuron fuction,including nerve function rescue,loss of nerve function and recording intracellular calciumsignal of neurons, and so on.