Preparation of the first national reference standard for determination of HEK293 cell DNA content / 中国生物制品学杂志

@#Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Expression and immunogenicity of glycoprotein H of pseudorabies virus in mammalian cells / 中国生物制品学杂志

@#Objective To express glycoprotein H(gH)of pseudorabies virus(PRV)in mammalian cells and detect its immunogenicity.Methods The gH gene fragment of PRV-XiangA strain was amplified by PCR and inserted into mammalian cell expression vector pIRES-neo3 to construct recombinant expression plasmid pIRES-gH,which was transfected to HEK-293F cells and cultured in suspension for 5 d. The cell culture supernatant was identified by Western blot and purified by nickel ion chromatography column. The purified gH was emulsified with ISA 201 VG adjuvant to immunize 8 female ICR mice,and 8 mice in control group were immunized with the same amount of adjuvant,which was strengthened at 5 and 8weeks after the first dose respectively. The blood samples were collected at 4,7 and 10 weeks after the first dose and detected for the titer of specific antibody and neutralizing antibody in serum of mice;The mice were challenged with PRVXiangA strain(1. 5 × 104TCID50)by nasal drops 2 d after the third blood collection,and observed for the morbidity and mortality daily.Results The recombinant expression plasmid pIRES-gH was constructed correctly as identified by sequencing. The gH protein was successfully expressed and modified by glycosylation in mammalian cells with good reactivity,and about 625 μg purified protein was obtained under 100 mL culture volume. After three times of immunization,mice produced high level of specific antibody and showed the effect of neutralizing PRV,and the titer of neutralizing antibody reached 1∶256. In the challenge test,all the mice in control group became ill and died,while half of the mice in gH immunized group did not get sick with a survival rate of 50%.Conclusion PRV gH was successfully expressed in mammalian cells,and its immune protection was confirmed for the first time,which provided experimental basis for the further research and application of gH,and also provided a new idea for the development of PRV subunit vaccine.

Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus / 生物工程学报

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O2 ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.

Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells / 生物工程学报

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

Expression and identification of recombinant human interferon λ1 in HEK-293F cells / 中国生物制品学杂志

@#Objective To express recombinant human interferon λ1(rhIFNλ1)by transient transfection in HEK-293F cells and identify it. Methods Two signal peptides[T cell receptor(TCR)and nature signal peptide(NSP)],three vectors[pcDNA3.4,ubiquitous chromatin opening element(UCOE)and PFR]and the target gene rhIFNλ1 were used to construct recombinant plasmids of six signal peptide-vector combinations. Using HEK-293F as host cells,the recombinant plasmids were transfected transiently to express rhIFNλ1 on a shake flask scale. The recombinant plasmid UCOE-Q46-λ with low glycosylation rhIFNλ1 was constructed by using NSP and vector UCOE,and transfected transiently into HEK-293F cells. The expressed product was purified by cation exchange chromatography(HiTrap SP FF),blue gel chromatography(HiTrap Blue HP)and gel filtration chromatography(Sephacryl S-100 HR),which was then analyzed by Western blot and reversed-phase HPLC,and determined for its molecular mass and N-terminal amino acid sequence by mass spectrometry. Results Six recombinant plasmids were constructed correctly as identified by double enzyme digestion and sequencing. With the extension of transfection time,the expression levels of the six expressed products increased gradually,and reached the highest level of10 ～ 20 mg/L at the 6th day of transfection. Double enzyme digestion and sequencing identification proved that the recombinant plasmid UCOE-Q46-λ with low glycosylation rhIFNλ1 was constructed correctly. The recombinant plasmid UCOE-Q46-λwas transfected into HEK-293F cells for 6 d. The purified product of cell culture supernatant showed a relative molecular mass of about 27 800 and a purity of 97. 372%,which showed specific binding to mouse anti-human IL-29/IFNλ1 monoclonal antibody;Two peaks were detected by reversed-phase HPLC,and the peak was showed at 15. 6 min and 20. 0 min respectively;The mass spectrometry molecular mass was about 24 000;The N-terminal five amino acids were G-P-V-P-T.Conclusion The rhIFNλ1 expressed by HEK-293F cells has high purity,which lays a foundation of further study of the protein.

The effect of HMS-01 on stably expressed hERG channel currents in HEK293 cells detected with the manualpatch clamp method / 药学实践杂志

Objective To test the cardiac toxicity of new compound HMS-01 and evaluate the safety profile for clinical trials. Methods Manualpatch clamp method was used to measure human Ether-a-go-go-Related Gene (hERG) potassium channel currents with different concentrations of HMS-01. Cisapride was selected as the positive control drug. HMS-01 was diluted to the concentration of 0.3, 1, 3, 10 and 30 µmol/L and applied to the cells. The changes in electrical currents were recorded and the inhibition rate was calculated. Results At the highest concentration of 30µmol/L, the inhibitory rate of HMS-01 on hERG channel was less than 30%. There was no obvious inhibitory effect compared with cisapride. Conclusion Compared with the cisapride, HMS-01 has no obvious inhibitory effect on hERG channel and has no cardiotoxicity.

Testosterona inhibe la actividad de la aldosterona sintasa silvestre y quimérica in vitro

Background: Familial hyperaldosteronism type I is caused by the generation of a chimeric aldosterone synthase enzyme (ASCE) which is regulated by ACTH instead of angiotensin II. We have reported that in vitro, the wild-type (ASWT) and chimeric aldosterone synthase (ASCE) enzymes are inhibited by progesterone and estradiol does not affect their activity. Aim: To explore the direct action of testosterone on ASWT and ASCE enzymes. Material and Methods: HEK-293 cells were transiently transfected with vectors containing the full ASWT or ASCE cDNAs. The effect of testosterone on AS enzyme activities was evaluated incubating HEK-cells transfected with enzyme vectors and adding deoxycorticosterone (DOC) alone or DOC plus increasing doses of testosterone. Aldosterone production was measured by HPLC-MS/MS. Docking of testosterone within the active sites of both enzymes was performed by modelling in silico. Results: In this system, testosterone inhibited ASWT (90% inhibition at five pM, 50% inhibitory concentration (IC50) =1.690 pM) with higher efficacy andpotency than ASCE (80% inhibition at five pM, IC50=3.176 pM). Molecular modelling studies showed different orientation of testosterone in ASWT and ASCE crystal structures. Conclusions: The inhibitory effect of testosterone on ASWT or ASCE enzymes is a novel non-genomic testosterone action, suggesting that further clinical studies are needed to assess the role of testosterone in the screening and diagnosis of primary aldosteronism.

Serum-Free Suspension Adaptation of HEK-293T Cells: Basis for Large-Scale Biopharmaceutical Production

Abstract Human Embryonic Kidney 293T cells (HEK-293T) are the most common host for viral vector production and are also widely employed for recombinant protein production. These cells are typically cultured in monolayer (adherent culture) using culture medium containing fetal bovine serum (FBS), which impairs batch-to-batch reproducibility and scale-up. The adaptation of adherent cell culture to suspension culture in chemically defined serum-free culture medium is an attractive approach for large-scale bioprocess implementation while aiming for a Good Manufacturing Practice (GMP) compliant production process. Therefore, in the present study, our goal was to adapt HEK-293T cells to serum-free suspension culture conditions and evaluate the feasibility of adapted cells to be transfected using different plasmid vectors for recombinant protein production. Firstly, the cells were efficiently adapted to serum-free conditions by sequential adaptation (FBS-containing medium weaning). During the whole process, parameters such as cell growth, viability and doubling time were evaluated and compared to the control (adherent serum-supplemented HEK-293T cell culture). Afterwards, these cells were adapted to suspension culture by using Erlenmeyer flasks in an orbital shaker platform, being able to achieve meaningful cell density with high viability. Adapted cells presented a transfection efficiency of approximately 50% for all vector constructs used (1054-GFP, Factor-VIII and Factor-IX). Overall, it was possible to successfully adapt HEK-293T cells to suspension and serum-free conditions, which represents an important step towards the development of a scalable and GMP-compliant production process. In addition, adapted cells efficiently expressed the different transgene tested, opening up possibilities for its use in recombinant protein production.

Construction of RHBDF2 gene over-expression lentivirus vector and establishment of RHBDF2 cell line stably expressing RHBDF2 / 吉林大学学报(医学版)

Objective: To construct the RHBDF2 gene over-expression lentivirus vector and to establish the KA. hy926 cells stably expressing RHBDF2, and to provide the evidence for the construction of RHBDF2 gene over-expression lentivirus vector and the establishment of RHBDF2 cells stably expressing RHBDF2. Methods: According to the sequence of RHBDF2 gene provided by NCBI, and the primers were designed and synthesized; the RHBDF2 gene was amplified by PCR method, and the target gene was cloned into the entry vector by Gateway cloning technology, and then subcloned into the lentivirus vector pLV [Exp]-EGFP to construct the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2; the lentivirus expression vector plasmid pLV I Exp]-EGFP and the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2 were co-transfected into the HEK293T cells with the virus-assisted packaging plasmids to package the lentivirus and the titer of the lentivirus was detected. The EA. hy926 cells infected with pLV [Exp]-EGFP-control were used as control group and the EA. hy926 cells infected with pLV [Exp]-EGFP-RHBDF2 were used as experiment group. The EA. hy926 cells stably expressing RHBDF2 were screened by puromycin. The fluorescent quantitative PCR (qPCR) and Western blotting methods were used to detect the expression levels of RHBDF2 mRNA and protein in the EA. hy926 cells in control group and experiment group. Results: The enzyme digestion electrophoresis and sequencing results showed that the gene sequence of the EA. hy926 cells over-expression lentivirus vector in experiment group was completely consistent with the designed and synthesized sequence. The lentivirus titer in control group was 1 X 10 TU • mL , and the lentivirus titer in experiment group was 3X 10 TU • mL . The EA. hy926 cells were successfully infected with the lentivirus under fluorescence microscope and the infection efficiency was above 95%. The qPCR detection results showed that the expression level of RHBDF2 mRNA in the EA. hy926 cells in experiment group was higher than that in control group (P'<0.01). The Western blotting results showed that the expression level of RHBDF2 protein in the EA. hy926 cells in experiment group was higher than that in control group ( P < 0. 05 ). Conclusion: The lentivirus vector over-expressing RHBDF2 is successfully constructed∗ and the EA. hy926 cell line stably up-regulating the expression of RHBDF2 is established by using pLV [Exp]-EGFP-RHBDF2 lentivirus.

CRISPR-Cas9-based site-directed knock-in of VEGF165 gene in a HEK293T cell / 国际生物医学工程杂志

Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system.

Establishment of a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in gene using CRISPR/Cas9 technique / 南方医科大学学报

OBJECTIVE@#To establish a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in gene using CRISPR/Cas9 technique.@*METHODS@#We designed 4 pairs of small guide RNA (sgRNA) for c.392G>T(p.131G>V) mutation site, and constructed exogenous PX458 plasmids expressing Cas9-sgRNA. The plasmids were transfected into HEK293T cells, and the cells expressing GFP fluorescent protein were separated by flow cytometry for further culture. After verification of the knockout efficiency using T7 endonuclease Ⅰ, the monoclonal cells were screened by limiting dilution and DNA sequencing to confirm the knockout. We detected the expressions of mRNA and protein and examined functional changes of the genetically modified cells.@*RESULTS@#We successfully constructed the Cas9-sgRNA exogenous PX458 plasmid based on the c.392G>T(p.131G>V) mutation site of gene. The editing efficiency of the 4 pairs of sgRNA, as detected by T7E1 enzyme digestion, was 6.74%, 12.36%, 12.54% and 2.94%. Sanger sequencing confirmed that the HEK293T cell line with stable knockout of c.392G>T(p.131G>V) was successfully constructed. The genetically modified cells expressed lower levels of mRNA and protein and showed reduced enzyme activity and proliferative capacity and increased apoptosis in response to vitamin K3 treatment.@*CONCLUSIONS@#We successfully constructed a stable HEK293T cell model with gene c.392G>T(p.131G>V) mutation site knockout to facilitate future study of gene repair.

Angiotensin Ⅱ induces apoptosis of HEK293T cells by up-regulating Cyr61 expression / 南方医科大学学报

OBJECTIVE@#To investigate the role of Cyr61 in angiotensin Ⅱ (AngⅡ)-induced functional changes in HEK293 cells and explore the mechanism.@*METHODS@#Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10 mol/L AngⅡ for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting.@*RESULTS@#The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells ( < 0.05). AngⅡ treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngⅡ treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group ( < 0.05). AngⅡ treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% (3.87 ± 0.83)%, < 0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngⅡ treatment ( < 0.05).@*CONCLUSIONS@#The up-regulation of Cyr61 expression is related with AngⅡ-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngⅡ-induced injury.

Establishment of HEK-293T cell model stably expressing TRPA1 channel / 中国药理学通报

Aim: To establish a HEK-293T cell line model stably expressing TRPA1 channel, and to verify the successful establishment of the model. Methods: The eukaryotic expression plasmid of TRPA1 was constructed and transfected into HEK-293T cells by liposome transfection method, the stable expression strain was screened by G418, and the transcription and protein expression of TRPA1 gene in HEK-293T cells were detected by RT-PCR and immunohistochemical techniques. Results: After restriction enzyme digestion and sequencing, it proved recombinant cloning plasmid of TRPA1 gene was successfully constructed; the results of PCR and immunohistochemistry showed that this recombinant plasmid could be transferred into HEK-293T cells with TRPA1 gene stable expression. Conclusions: A HEK-293T cell line with stable expression of TRPA1 channel is successfully constructed, which lays the foundation for studying the physiological and pathological functions of TRPA1 in vitro and screening of relevant TR-PA1 channel regulators.

Construction and identification of miR-186 overexpression lentiviral vector / 吉林大学学报(医学版)

Objective: To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and to explore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods: The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoR I and Age I restriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results: The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×108 TU · mL-1 and 5 × 108 TU · mL-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12. 640 0 ± 0. 788 4) was significantly increased by 15. 07 times (t=14. 72, P<0. 01) compared with control group (0. 838 7 ± 0. 145 6). Conclusion: The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus successfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.

Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via L-arginine deprivation in HEK-293 cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Mycoplasma infection is the most prevalent contamination in cell culture. Analysis of cell culture in laboratories from different countries shows that mycoplasma contamination ranges from 15% to 80% and, in some cases, even reaches 100% (Chernov et al., 2014). Whilst mycoplasma infection is not visible to the naked eye in cell culture, the consequences of mycoplasma contamination have been shown to induce a number of cellular changes, for example, increased resistance to chemotherapeutic drugs. Therefore, any results obtained from tissue culture studies, in the presence of mycoplasma contamination, potentially render the data invalid (Kim et al., 2015; Gedye et al., 2016). As such, mycoplasmas are not harmless bystanders and cannot be ignored in in vitro studies.

Expression of fluorescent tagged recombinant erythroferrone protein

Objective: To produce fluorescent tagged recombinant erythroferrone protein (ERFE-eGFP) for laboratory investigations. Methods: Erythroferrone (ERFE) gene was fused to green fluorescent protein (eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5 α and the eGFP-fused ERFE (ERFE-eGFP) protein was expressed in human embryonic kidney (HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE-eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE-eGFP fusion protein (approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE-eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.

The regulation and gating mechanism of Cav1.2 current by KCNE1 subunit / 中华急诊医学杂志

Objective To explore the regulation and mechanism of Cav1.2 current by KCNE1. Methods Transient transfection was used to transfer Cav1.2 channel plasmids separately or together with KCNE1 plasmids into HEK293 cells. The experiment was divided into 2 groups (15 cells in each group):Cav1.2 group, Cav1.2+KCNE1 group.The whole-cell patch clamp technique was used to record Cav1.2 current and gating dynamics. Results After co-transfection of KCNE1 with Cav1.2, Cav1.2 current decreased significantly. At 0 mV, peak current density of Cav1.2 was reduced from (-14.8±2.5) pA/pF to (-7.5±1.6) pA/pF (n=15, P<0.01). Based on the gate control mechanism, it is found that the regulation of Cav1.2 current by KCNE1 mainly makes the steady-state inactivation curve of the channel shifted to a more negative direction, thus accelerating the inactivation. Meanwhile, the recovery process of the channel after inactivation is slowed down and the recovery time constant was prolonged. Conclusions The KCNE1 subunit can reduce the current density of Cav1.2 by changing the channel inactivation and recovery process.

Targeting knockout of DMD gene exon51 in HEK293T cell based on CRISPR/Cas9 system / 基础医学与临床

Objective To knockout the exon51 of DMD gene in HEK293T cells using the CRISPR/Cas9 system. Methods Design the target sequences of sgRNA and clone them into plasmid PX459 respectively; transfer these plasmids into HEK293T cell and extract the total genome DNA; test the activity of sgRNAs with surveyor assay, choose the most efficient one in each end;construct plasmid PX459-2sgRNA and transfer it into HEK293T cells;check whether the exon51 has been knocked known with PCR and T vector sequencing. Results 50% of HEK293T cells' DMD gene exon51 were knocked out,showing a high gene editing efficiency. Conclusions We successfully establish a platform to target knockout the exon51 of DMD gene and provide an important experimental basis for the treatment of DMD and other genetic diseases.

The mechanism of QDPR on reducing apoptosis induced by fatty acids / 安徽医科大学学报

Objective To investigate the effects of dihydropteridine reductase (QDPR) on regulating apoptosis induced by plamitic acid(PA). Methods The transfection of HEK293T cells experiment was divided into 3 groups. A group was the control vector group. B group was the control vector group induced by PA. C group was the recombinant plasmid QDPR group induced by PA. First, control vector and recombinant plasmid QDPR was respectively transfected into HEK293T cells. After 24 h, PA with concentration of 0. 5 mmol/L was added into the medium of above cells. The cells of control vector group, the cells of control vector group induced by PA and the cells of recombinant plasmid QDPR group induced by PA were cultured for another 24 hours. At last, cells were harvested to detect tetrahydrobiopterin (BH4) and reactive oxygen species(ROS) generation, Beclin 1, Caspase 3 and Beclin 2 expression. Results ① After transfection, the recombinant plasmid QDPR was successfully constructed and expressed in cells.② There was no significant difference between A group and B group in BH4 generation. Compared with B group, BH4 generation increased in C group (P < 0. 05).③ ROS generation was increased in B group compared with A group, and decreased ROS generation in C group compared with B group (P < 0. 05).④ Western blot analysis revealed that Beclin 1 and Caspase 3 increased (P < 0. 05 ) while Beclin 2 decreased in B group compared with A group (P < 0. 05). Compared to B group, Beclin 1 and Caspase 3 decreased while Beclin 2 increased in C group (P < 0. 05 ). Conclusion QDPR may regulate apoptosis induced by fatty acids by decreasing the generation of ROS and increasing the level of BH4 and the expression of Beclin 2 associated with anti-apoptosis.

Effects of Daurisoline on L-Type Calcium Channel CaV1.2 Expressed in HEK293 Cells / 中国药师

Objective:To investigate the effects of daurinsoline (DS) on L-type calcium channel Cav1.2 expressed in HEK293 cells.Methods:Cav1.2 was transferred into HEK293 cells using Lipofectamine 2000, and the effects of DS on Cav1.2 currents (ICav1.2) were analyzed by whole-cell patch clamp techniques. Results:DS at 1,3 and 10 μmol·L-1could inhibit the ICav1.2in HEK293 cells in a dose-dependent manner. The inhibitory rate was(14.68 ± 4.02) %,(32.37 ± 6.63) % and(59.63 ± 5.23) %,respectively. The inhibitory rate of DS at 3 μmol·L-1was 40 % of that of 3 μmol·L-1isradipine(a L-type calcium channel blocker). Conclusion:DS can inhibit the ICav1.2in HEK293 cells in a dose-dependent manner and the inhibition of DS is weaker than that of isradipine.