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A cell co-culture method for the analysis of synapse formation

A technology of nerve synapses and neuron cells, applied in the field of bioengineering, can solve problems such as high cost, complicated operation, and long time consumption, and achieve the effect of convenient operation

Inactive Publication Date: 2018-07-13
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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Problems solved by technology

[0005] When screening genes in this co-culture method, if dozens or even hundreds of genes are screened in parallel at one time, HEK293T cells in a 24-well plate are digested and added to neuronal cells in a 24-well plate. Contamination, the HEK293T cells in each well need to be replaced during operation, the operation is complicated, time-consuming, and relatively high in cost

Method used

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Embodiment Construction

[0015] The present invention will be described in detail below in conjunction with specific examples, but the protection scope of the present invention is not limited to the following examples.

[0016] The specific operation method in the present embodiment is as follows:

[0017] (1) Mouse hippocampal neuron cells were cultured in a 24-well plate until day 8, and 20,000 subcultured HEK293T cells were added to each well, shaken gently, and placed back in a constant temperature incubator for 24 hours.

[0018] (2) In the ultra-clean workbench, take a sterile 1.5mL EP tube, add 100uL serum-free medium Opti-MEM, then continue to add 0.15ug GFP plasmid and 0.5ug Neuroligin2 (NL2) plasmid in sequence, and flick it with your fingers Centrifuge the tube wall for 5-6 times to fully mix the GFP plasmid and NL2 plasmid in Opti-MEM. Finally, add 2ug polyethyleneimine (PEI), flick the wall of the centrifuge tube 5-6 times with your fingers to fully mix the PEI and the two plasmids, then...

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Abstract

The invention provides a cell co-cultivation method used in the analysis of synapse formation, comprising the following steps: adding subcultured non-neuronal cells, namely HEK293 T cells, to 24-well primary cultured neuronal cells in an in vitro environment Medium, mixed and cultured for more than 24 hours; PEI transfection method was used to transfect HEK293 T cells, and the exogenous genes transfected in each well were different, and the transfected HEK293 T cells overexpressed exogenous genes, Under these conditions, co-culture was carried out for more than 48 hours; the presynaptic marker protein synapsin was immunostained to determine whether a synaptic structure was formed in each well. The co-cultivation method provided by the present invention solves the deficiencies in the prior art. The method is simple and convenient to operate, relatively low in cost and short in time, can screen genes well and verify gene functions, and can be widely used in laboratories .

Description

technical field [0001] The invention provides a cell co-culture method, in particular a cell co-culture method for screening genes involved in central nervous system synapse formation, and belongs to the technical field of bioengineering. Background technique [0002] A synapse is the contact between two neurons. The method for screening new genes involved in synapse formation in the central nervous system is mainly to use subcultured non-neuronal cells and primary cultured neuronal cells to co-culture. [0003] Under normal physiological conditions, co-culture of subcultured non-neuronal cells and primary cultured neuronal cells will not form synapses. When genes involved in synapse formation are overexpressed in subcultured non-neuronal cells, the The co-culture of cultured neurons results in the formation of synaptic structures. [0004] At present, the commonly used method of co-cultivation is to first overexpress the exogenous gene in HEK293 or COS7 cells subcultured ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793C12N5/071G01N33/533
Inventor 阳小飞蒋伟陈健王明明龚吉红梅颖
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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