This study aims to construct the recombinant lentivirus vector containing specific small interfering RNA (siRNA) targeting rat CREB binding protein(CBP)gene and to identify its function of inhibiting the expressions of acetylated histone in primarily cultured hippocampal neurons. Firstly, we constructed four kinds of recombinant lentivirus siCBP. And then we used them to infect the primarily cultured hippocampal neurons, and performed real-time PCR, western blot respectively to detect the expressions of CBP. Afterwards, the most effective lentivirus siCBP was used to infect the primarily cultured hippocampal neurons, and then the HAT activity and protein expressions of acetylated histone Ac-H3, Ac-H4 of the neurons were examined. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in lentivirus vector. Besides, CBP mRNA and protein expressions in neurons were found to be with varying degrees of decreases after infections of the four kinds of lentivirus siCBP. Furthermore, the representative and most effective lentivirus GR806 could effectively inhibit the HAT activity and the protein expressions of Ac-H3, Ac-H4 in neurons. It provides the experimental basis for the subsequent application of siCBP to clarify the effects and corresponding molecular mechanism of the CBP-dependent histone acetylation on learning and memory function in hippocampus.
Acetylation ; Animals ; CREB-Binding Protein ; metabolism ; Genetic Vectors ; Hippocampus ; cytology ; Histones ; metabolism ; Lentivirus ; Memory ; Neurons ; metabolism ; Primary Cell Culture ; RNA, Messenger ; RNA, Small Interfering ; Rats ; Real-Time Polymerase Chain Reaction

Objective:To analyze the correlation between cuproptosis genes and immune infiltration during the occurrence and development of multiple sclerosis(MS),and to predict the traditional Chinese medicine,to provide theoretical basis for the mecha-nism study of cuproptosis in MS immune infiltration and Chinese medicine to intervene in immune regulation.Methods:The gout chip of MS was downloaded from the GEO database and standardized;based on the processed data,immune cells and functions were ex-tracted and quantified,and the correlation and differences of immune cells and functions were analyzed;at the same time,cuproptosis genes related to MS were screened out,a risk model was constructed,and enrichment analysis was carried out;the prediction of cu-proptosis genes and immune-related biological processes were carried out.Results:① Among immune cells,T follicular helper cell and B cell showed the strongest positive correlation;among the immune functions,parainflammation and typeⅠIFN reponse showed the strongest positive correlation;②B cell,T helper cell and human leukocyte antigen were lowly expressed in MS patients,while major histocompatibility complex class Ⅰ,parainflammation and typeⅠIFN response were significantly expressed;③ The cupropto-sis genes associated with MS were SLC31A1,PDHA1,NLRP3,MTF1,GLS and DBT,of which DBT was the most likely risk factor for MS;④The occurrence and development of MS involves biological processes such as IL-4 production,T-helper cell differentiation,and acute inflammatory response,which were related to pathways such as arginine biosynthesis,citrate cycle,and propanoate metabo-lism;⑤Banxia,Danshen,Jianghuang and other traditional Chinese medicines may be used as potential molecular drug sources.Con-clusion:The expression of cuproptosis gene is closely related to MS-related immune cells and functions,which can provide support for the basic research of MS.

Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats -associated protein (CRISPR/Cas) has been developed as a precise, efficient, affordable and sensitive nucleic acid detection tool due to its efficient targeted binding ability and programmability. At present, biosensors based on CRISPR-Cas system have shown excellent performance in the detection of nucleic acid of pathogens, which has attracted widespread attention, and is expected to replace the conventional detection methods. This review summarizes the latest research progress of biosensors based on CRISPR/Cas system for detecting nucleic acid of pathogens.
Biosensing Techniques ; CRISPR-Cas Systems/genetics* ; Nucleic Acids/genetics*