The Screen Of Cell Adhesion Molecules

The human brain, which consists hundreds of billions of neurons, is one of the most important organs in human body. Neurons translate information by contacted with each other, realizing the stimuli and conducting function of nervous system. The information translation between neurons is accomplished by synapses. The synapse is the basic structural and functional unit of the nervous system. There are about1015 synapses in the brain. Synapses are composed by three portions: presynaptic,synaptic cleft and postsynaptic. Synaptic cleft is a narrow( ? 20 nm) layer of extracellular space with various proteins and carbohydrates. The most important function of synaptic cleft is keeping the presynaptic and postsynaptic link to complete the transmission of information by the interaction of proteins, which locate at the synaptic cleft and are called cell adhesion molecules(CAMs). CAMs contain three domains: an intracellular domain that interacts with the intracellular scaffold protein,a transmembrane domain and an extracellular domain that interacts with other CAMs.Most of CAMs localize at the center of the synapse, whereas others are located at the outer rims of pre-synaptic active zones and post-synaptic regions. CAMs have multiple functions at synapses. They can recognise the interaction site of pre/postsynaptic membrane, translate information, help the synaptic vesicle release and also play an essential role in promoting synapse formation and maturation,maintaining synapse number and type, accumulating neurotransmitter receptors and ion channels, controlling neuronal differentiation, and even regulating synaptic plasticity directly. The abnormal expression of CAMs could results in many neurological disorders. At present, more and more persons are suffering from the diseases of nervous system. The diseases of nervous system have became one of the most horrible killers of human health. It is very important to find more CAMs and understand their function in the pathogenesis of some neurological disorders. Some CAMs are found according the researches, but there should be more remain unknown comparing with the vast number and various synapses. Studies of synapses have shown that CAMs, which express in non neuronal cells, can induce synaptic accumulation. Synapses may not be eliminated by any of CAM molecule deletion, but the structure and function of synapse could be damaged by the disfunction of CAMs,and even worse, some neurological disorders may be induced. So, we seed the non neuronal cells to the neurons, in order to find more CAMs that could induce thesynaptic accumulation. We also plan to identify the functions of those molecules we found by overexpression or suppression techniques. More CAMs can be uncovered through our researches.We screened more than 16000 genes of human. The gene library is made by Broad laboratory in MIT University and other well-known institutions, called CCSB-Broad lentivirus library. It bases on the ORF eome8.1, which was made by the same organization earlier. With the LR interaction of Gateway Clone, the target gene ORF is transferred into Plx304-Blast-v5 vector. Nowadays, Gateway Cloning Technology is one of the most advanced cloning technologies with high efficiency in the world. Because there are 222 genes, which are larger than 3Kb, lacking in the CCSB-Broad lentivirus library, we first cloned them into the vector pCDNA3.2V5-DEST with the help of LR Clonase? enzyme mix, which contain the recombinant factor Xis/IHF/Int. The lethal gene ccdB was changed by the target gene during the cloning. In the enzyme digestion, we only used restriction enzyme BglII.This method can increase the efficiency and save money as well. We got all the right samples of the LR Clones with the identification of enzyme digestion. To test whether all the plasmid can express the protein, we used the entire protein to confirm by western blot. We found that the highest efficiency can be observed when HEK293 T cells cultured in 3.5 cm dishes were transfected by 4?g plasmids. Now we have found110 plasimds could express membrane proteins.We get 16394 human genes by LR clone and we can screen the genes by a stable method. We co-transfected 1.5?g target gene and 0.5?g GFP-FUGW into HEK293 T by PEI. 24 hours later, seed the HEK293 T into hippocampal neurons, which have been cultured for nine days. 36 hours later, we performed immustaining experiments and observed the images with high intension of camera system. A ring like structure close to the HEK293 T cell can be observed if the target gene is involved in synapses formation. In our experiments, we found that only 0.25?g positive gene can cause significant synapse aggregation. Thus, for increasing the efficiency, we mixed 6different but equal concentration plasmids and transfected the mixture into HEK293 T cells together. Coculture Index(C.I.) was used to represent the aggregation degree of synapses. It is quantized by the fluorescence intensity of synapses. For easier judging whether a target gene is a CAM, we set a ring area that starts from the edge of HEK293 T cell and 2 ?m for both inner and outer radius are chosen to count C.I. There is significant aggregation observed if C.I.>1. With this method, we have found 4positive mix plasmids having potential roles in synapses formation.