Objective:To construct the over-expressed lentivirus vector of PRDM5 gene and establish the Neuro-2a cells stably transfected PRDM5,and to provide the basis evidence for exploring the effect of PRDM5 in pathogenesis of ischemic stroke(IS).Methods:The sequence of PRDM5 was searched and designed based on NCBI.The PRDM5 gene was amplified by PCR and ligated with the lentiviral vector GV492 digested by BamH Ⅰ and Age Ⅰ restriction enzymes to form the GV492-PRDM5 over-expression recombinant plasmid.The positive clones with similar length and size to the target gene fragment were screened by PCR and sent to Shenggong Bioengineering(Shanghai)Co.Ltd.for identification.The correctly-sequenced GV492-control plasmid and GV492-PRDM5 over-expression recombinant plasmid were transfected into the HEK293T cells,respectively.After 48 h of transfection,the lentiviruses were collected by centrifugation,and they were GV492-control lentivirus and GV492-PRDMS over-expression lentivirus;the titers of these two lentiviruses were determined by lentiviral titer assay.The Neuro-2a cells were divided into GV492-control group and GV492-PRDM5 group,and then infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus,respectively,with a lentivirus multiplicity of infection(MOI)of 100.The Neuro-2a cells successfully infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were screened with puromycin(10 mng-L-1)after 72 h of infection.The growth status and the expression of green fluorescence protein of Neuro-2a cells in GV492-control group and GV492-PRDM5 group were observed by fluorescence microscope.The expression levels of PRDM5 mRNA and PRDM5 protein in the Neuro-2a cells in two groups were detected by real-time fluorescence quantitative RCR(RT-qPCR)and Western blotting methods.Results:The PCR results showed that the length of the positive transformant of GV492-PRDM5 recombinant plasmid was about 684 bp,and the gene sequence of GV492-PRDM5 over-expression recombinant plasmid was consistent with the designed and synthesized PRDM5 over-expression sequence.The titers of GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were both 2.5×108TU·mL-1 The Neuro-2a cells in GV492-control group and GV492-PRDM5 group grew well,and the expressions of green fluorescence protein were found under fluorescence microscope.The RT-qPCR results showed that the expression level of PRDM5 mRNA in the Neuro-2a cells in GV492-PRDM5 group was significantly increased compared with GV492-control group(P＜0.01).The Western blotting results showed that the specific bands appeared in the Neuro-2a cells in GV492-control group and GV492-PRDM5 group with a relative molecular weight of 75 000;compared with GV492-control group,the expression level of PRDM5 protein in the Neuro-2a cells in GV492-PRDM5 group was increased(P＜0.01).Conclusion:The over-expression lentivirus vector of PRDM5 gene is successfully constructed,and the stably transfected GV492-PRDM5-Neuro-2a cells are established.

Objective:To construct the eukaryotic cell translation initiation factor 4A3(EIF4A3)short hairpin RNA(shRNA)lentiviral vector,and to establish the Neuro-2a-EIF4A3-shRNA stable transfection cell line.Methods:The EIF4A3 gene sequence was retrieved from the National Center for Biotechnology Information(NCBI)database;the PCR identification primers were designed and synthesized,and connected to the lentiviral GV493 vector digested with Eco R I and Age I enzymes to construct the GV493-EIF4A3-shRNA lentiviral plasmid;PCR method was used to screen the positive clones,which were sequenced for the identification;the GV493 empty plasmid and GV493-EIF4A3-shRNA recombinant plasmid were transfected into the HEK293T cells,regarded as GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus,respectively.After 48 h of transfection,the lentiviruses were collected for packaging and the viral titer was determined.The Neuro-2a cells were divided into blank group,GV493 control group,and GV493-EIF4A3 shRNA group.The Neuro-2a cells in blank group were untreated,and the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group were infected with the respective lentiviruses at a multiplicity of infection(MOI)of 100.The infected Neuro-2a cells were selected by 10 mg·L-1 puromycin,and the growth status and green fluorescence expression of the Neuro-2a cells in various groups were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of EIF4A3 mRNA and protein in the Neuro-2a cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the GV493-EIF4A3-shRNA recombinant plasmid was consistent with the designed EIF4A3-shRNA sequence,indicating successful construction of the GV493-EIF4A3 lentiviral vector.The fluorescence microscope observation results showed that there was strong fluorescence expression and good growth status in the HEK293T cells,confirming successful lentiviral packaging.The viral titers for GV493 control lentivirus and GV493-EIF4A3-shRNA lentivirus both were 2×108 TU·mL-1.The growth status of the Neuro-2a cells in GV493 control group and GV493-EIF4A3 shRNA group was good,and they expressed green fluorescence,indicating successful construction of the stable transfection cell line.The RT-qPCR results showed that compared with blank group and GV493 control group,the expression level of EIF4A3 mRNA in the cells in GV493-EIF4A3 shRNA group was significantly decreased(P<0.01).The Western blotting results showed that the specific bands was at a relative molecular mass of 49 000,indicating successful EIF4A3 protein expression in the Neuro-2a cells.Compared with blank group and GV493 control group,the expression level of EIF4A3 protein in the cells in GV493-EIF4A3 shRNA group was significantly decreased(P<0.01).Conclusion:The GV493-EIF4A3-shRNA lentiviral vector is succfssfully constructed,and the Neuro-2a-EIF4A3-shRNA stable transfection cell line is established;the results provide the reference for the study of the effect of EIF4A3 on the intracranial atherosclerosis.

Objective:To construct an over-expression lentiviral vector of the dedicator of cytokinesis 4(DOCK4),and to establish DOCK4 stably over-expressing Neuro-2a cells.Methods:The DOCK4 sequence was searched in the National Center for Biotechnology Information(NCBI)and primers were designed and synthesized;polymerase chain reaction(PCR)method was used to amplify the DOCK4 gene sequences.After digestion with BamH Ⅰ and Age Ⅰ restriction endonucleases,the DOCK4 gene sequences were ligated with the digested lentiviral vector GV492 to construct the GV492-DOCK4 over-expression recombinant plasmid.The positive clones with a similar length to the target gene fragment were screened and identified by PCR method.The GV492-control plasmid and GV492-DOCK4 over-expression recombinant plasmid were transfected into the HEK293T cells,and the lentivirus was collected and titered 48 h after transfection.The Neuro-2a cells were divided into GV492-control group and GV492-DOCK4 group,and the cells were infected with GV492-control lentivirus and GV492-DOCK4 over-expression lentivirus,respectively,and the multiplicity of infection(MOI)was 100.After 72 h of infection,the successfully infected Neuro-2a cells were screened by using puromycin(10 mg·L-1).The growth status of Neuro-2a cells and the expression of green fluorescent protein in various groups were observed under fluorescence microscope.Real-time quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of DOCK4 mRNA and DOCK4 protein in the Neuro-2a cells in various groups.Results:The PCR results showed that the gene fragment length of the GV492-DOCK4 over-expression recombinant plasmid was approximately 691 bp.The sequencing results showed that the gene sequence of the GV492-DOCK4 over-expression recombinant plasmid was consistent with the designed over-expression sequence of DOCK4.The titers of the lentiviruses in GV492-control group and GV492-DOCK4 over-expression group were 2.5×108 TU·mL-1 and 2.5×108 TU·mL-1,respectively.The fluorescence microscope observation results showed that Neuro-2a cells in various groups grew well and expressed green fluorescent protein.The RT-qPCR results showed that compared with GV492-control group,the expression level of DOCK4 mRNA in the Neuro-2a cells in GV492-DOCK4 group was significantly increased(P＜0.01).The Western blotting results showed the specific bands near the relative molecular mass of 225 000 in various groups.Compared with GV492-control group,the expression level of DOCK4 protein in the Neuro-2a cells in GV492-DOCK4 group was significantly increased(P＜0.01).Conclusion:This study successfully constructs the DOCK4 over-expression lentiviral vector and establishes the Neuro-2a cells stably over-expressing DOCK4.

Although different types of drugs are available for postmenopausal osteoporosis, the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis agents. Here, we defined that norlichexanthone (NOR), a natural product, is a ligand of estrogen receptor-alpha (ER