Influence Of Conditional Knockout Of Prkcb Gene In Microglia On Depression-like Behavior In Mice

Objective:Major depressive disorder is a highly heterogeneous psychiatric disorder and its etiology remains unclear.Recently,much attention has been paid to the"inflammatory hypothesis",which suggests that inflammatory responses are involved in the pathogenesis of depression.Microglia,the key immune cells of the central nervous system,are the main mediators of many inflammation-related diseases.Whereas protein kinase C（PKC）plays an important role in regulating microglia activation,abnormal expression of PKC and Prkcb which is the gene encoding PKC,is also present in depressed patients.Therefore,this study aimed to investigate the effect of conditional knockout of Prkcb gene in microglia on depressive-like behavior in mice,and thus to explore whether Prkcb is a key gene in the occurrence of depressive-like behavior in mice.Methods:Part I Construction,propagation,and identification of conditioned knockout mice with Prkcb gene in microglia Using Crispr/Cas9 gene editing technology to construct mice that conditionally knock out the Prkcb gene from microglia,conducting subsequent mouse reproduction through selfing or hybridization,and using Polymerase Chain Reaction（PCR）to identify mouse genotypes;Part II Behavioral tests on mice with the Prkcb gene conditionally knocked out of microglia 14 mice that have undergone conditional knockout of the Prkcb gene in microglia and 14 mice that have not undergone conditional knockout of the Prkcb gene in microglia were select as the control group mice were select as the experimental group mice（all of them were 6-8week old mice with similar appearance）.Behavioral tests on the selected mice were conducted,such as the sucrose preference test,open field test,novel object recognition test,light dark box test and tail suspension test.Behavioral experiments were used to explore the depression-like behaviour in mouse model with the Prkcb gene conditionally knocked out of microglia.Results:Part I The PCR genotype identification results showed that there were 14 mice with genotype Prkcb^-/-:Cre⁺,and the included mice serial numbers were:1,2,3,4,9,12,14,17,18,19,21,22,23,28.We have constructed mouse model with conditional knockout of the Prkcb gene in microglia successfully;Part II In the sucrose preference test,there was no statistical difference in the percentage of sucrose preference between control and experimental mice（P>0.05）;in the open-field test,there was no statistically significant difference in the total distance,duration in the central compartment and counts of entering the central compartment between control and experimental mice in the first 10minutes,the second 10 minutes and the third 10 minutes（all P>0.05）;in the novel object recognition test,there was no statistical significance in the difference score（novel object interaction–familiar object interaction）and discrimination ratios of the control between the control and experimental groups（all P>0.05）;in the light dark box test,there was no statistically significant difference in the frequency of transfer from the light box to the dark box or from the dark box to the light box between the control and experimental groups（all P>0.05）;in the tail suspension experiment,there was no statistically significant difference in the resting time between the control and experimental groups（P>0.05）.Conclusion:The mice that conditionally knockout Prkcb gene in microglia were successfully constructed by Crispr/Cas9 genome editing technology.Additionally,knocking out the Prkcb gene alone is not sufficient to induce depressive behavior in individual.