Preliminary Study On The Expression And Distribution Of TMC7 MRNA In The Nervous System

The transmembrane channel-like gene family(TMC)encodes a group of transmembrane proteins with eight transmembrane regions(TM1-TM8),and the family includes eight members TMC1-TMC8.Their conservation in the evolutionary process indicates that these genes have important cellular functions.However,due to TMC proteins are difficulty to express in exogenous cell,lack of effective and specific antibodies,and lack of the protein structures,the research on the expression and function of the TMC protein family has been difficult to break through.Recent studies have found that mouse TMC1 and TMC2 proteins are expressed in the mechanically sensitive ion channels of the cochlear hair cells,which is an important basis for the inner ear hair cells to sense mechanical stimuli and transform it into nerve impulses during auditory process.This study suggests that transmembrane channel-like gene family members may be ion channel proteins and play an important role in animals' perception of external stimuli.According to the database and our previous studies,it was found that TMC7 mRNA is expressed at a higher level in the rat nervous system and peripheral primary sensory ganglia DRG,suggesting that TMC7 may play a important role in the perception external stimuli from environment in sensory nervous system.To prove this hypothesis,we establish TMC7 knockout rats by the CRISPR/cas9 and systematically studied its phenotype to determine the possible physiological functions of TMC7 protein.On the other hand,the TMC7 knockout rats provide a good control for the study of TMC7 gene expression in the nervous system.Because there is still a lack of specific antibodies against TMC7 protein,the focus of this paper is to use TMC7 knockout rats as controls to explore the expression and distribution of TMC7 mRNA in the central nervous system and peripheral nervous system(especially DRG neurons)through Basecope and RNAscope methods,which are two unique types of in situ hybridization techniques.This study can enhance people's understanding of the expression of TMC7 in the nervous system.Part 1 Study on the expression and distribution of TMC7 gene in brain and DRGObjective: To confirm the gene knockout result of TMC7 gene knockout rats,and to analyze the expression and its distribution characteristics of TMC7 gene in the central nervous system and the peripheral nervous system.Methods:(1)Using CRISPR/cas9 system to knockout TMC7 gene in Rat.Extract DNA from rat's tail for genotype identification and confirm the gene knockout result.And RT-PCR was also used to verify the of expression TMC7 in knockout rat DRG and col.(2)Using TMC7 knockout rats as control,the expression and distribution of TMC7 mRNA in the central nervous system of WT rats was detected by Basescope.(3)Through Basescope experiment to observe the expression and distribution of TMC7 mRNA in DRG of WT rats and knockout rat.Results:(1)About ten days after birth,all the rats were marked by toe cutting,and genomic DNA was extracted from tail of rat.Transgenic rat identification was done using PCR technique followed by gel electrophoresis.The target band of TMC7 was about 620 bp,while the band of WT was about 466 bp,rats with homozygous genotypes can be used as the experimental group and WT mice as the control group.(2)Basescope experiment result on brain shows that there is rich expression of TMC7 mRNA in brain of WT rat and the expression was almost gone in TMC7 konckout rat.TMC7 mRNA is widely distributed in the central nervous system with certain variaty in different brain regions: The expression rate was 22.43% in the hippocampus,34.55% in the striatum,40.04% in the dorsal raphe nucleus,40.74% in the substantia nigra,47.44% in the prelimbic cortex,and 34.21% in the ventral tegmental area of the midbrain.The cerebellum area was 17.13%.This result suggests that TMC7 is expressed more in the midbrain and prefrontal cortex than other regions.(3)Basescope experiment results on peripheral sensory ganglia DRG shows that there is rich expression of TMC7 mRNA in DRG of WT rat and the expression was gone in TMC7 konckout rat.It is worth notcing that TMC7 is more expressed in small diameter neuronal cells than it does in large diameter nerves,which suggests that it may be a relationship between the TMC7 and the physiological regulation activities of small-diameter neurons.Conclusions: TMC7 mRNA is widely distributed in the brain.The abundance of TMC7 mRNA is different in different brain regions and is relatively high in the midbrain and prefrontal cortex.TMC7 mRNA is also widely distributed in DRG,especially in small neurons,suggesting that it may be related to the functional activities of small-diameter neurons.Part 2 Characteristic and distribution analysis of TMC7 mRNA in DRGObjective: At the transcript level,we observe the expression of the TMC7 gene and other various classification marker genes,subsequently,to investigate the characteristic distribution of TMC7 mRNA in DRG neurons and to speculate their related functions.Methods:(1)RNAscope technology was used to observe the co-expression of TMC7 and DRG sensory marker genes such as Nav1.8,TRPV1,CGRP and NEFH.(2)In combination with RNAscope and immunofluorescence methods,TMC7 was labeled with an mRNA probe,and Piezo2 was labeled with a protein antibody.Finally,the fluorescence distribution of these two genes on DRG cells was observed.(3)The single cell PCR technique was used to detect the co-expression of the TMC7 mRNA and various other classification marker genes at the single cell level.Results:(1)RNAscope results showed that the percentage of cells expressing Nav1.8,CGRP,TRPV1,and NEFH mRNA in TMG7 mRNA-positive DRG neurons was 62%,18.7%,51.3%,and 15%,respectively.It is suggested that the physiological function of TMC7 may be more related to Nav1.8 and TRPV1.(2)Combination of the two methods showed that here was higher co-labeling among Piezo2 and TMC7,suggesting that there may be close connection between them.(3)Using single cell PCR technology,the expression of TMC7 mRNA was observed at the single cell level of DRG.According to the distribution of cell diameter,it was found that TMC7 was relatively expressed in 15-50 ?m small-to-medium-diameter DRG neurons,which is consistent with the results of the first part of Basecope.By observing the co-expression of TMC7 mRNA with other DRG neuron marker genes,it was found that TMC7 mRNA seems to co-express more with GAT1,Sst,Mrgprb4,and TRPV1,but less co-expression with NEFH.Conclusions: The above experiments demonstrated that TMC7 mRNA co-expressed with Nav1.8 and TRPV1 was relatively high,but co-expression with CGRP and NEFH was less,and there was a close relationship between TMC7 and Piezo2.The results of single cell PCR also confirmed these points.These results suggest that TMC7 may be involved in the conduction and regulation of pain in the nervous system.