Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Expression, purification of recombinant human cryptochrome I and its application in preparation of protective agent for radiotherapy / 生物工程学报

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.

Immunological efficiency induced by HIV-1 p24 DNA combined with P24 protein / 生物工程学报

New strategies to improve vaccine efficacy against human immunodeficiency virus type 1 (HIV-1) are still required. DNA vaccines, exhibiting potential advantages over conventional vaccines for their simplicity and versatility, can induce specific humoral and cellular immune responses. We developed a recombinant pVAX1 DNA vector carrying p24 gene of HIV-1. The results showed that pVAX1 mediated gene possessed the ability of effective expression in both transfected 293T cells and BALB/c mice. And pVAX1-p24 DNA prime and boost immunization can induce significant P24-specific humoral immune responses and cellular immune responses in BALB/c mice. Furthermore, immunization with pVAX1-p24 DNA prime and protein boost induced 7.3 to 8.0-fold greater p24-specific humoral responses than pVAX1-p24 DNA prime and boost, while the cellular immune responses induced by combined immunization was lower. The results suggested that pVAX1-p24 DNA and P24 protein vaccine is a promising HIV-1 vaccine, and the selections of the immunization strategies are important for the immunization results.

Silencing suppressors: viral weapons for countering host cell defenses

RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors.

Polyethylenimine and minicircle DNA based gene transfer / 生物工程学报

Polyethylenimine (PEI) is one of the most characterized non-viral vectors. It can condense DNA in a good manner and achieve high transfection efficiency. Minicircle DNA (mc-DNA) is a novel kind of supercoiled DNA which is devoid of bacterial backbone. mc-DNA is superior to conventional DNA for its higher transfection efficiency and longer time-span. In this study, we combined PEI and mc-DNA in gene delivery system. We investigated the physicochemical and biochemical effects of this non-viral system and further explore its potential in tumor gene therapy. mc-DNA was obtained by recombination of parental plasmid in the presence of L-arabinose, and complexed with PEI. The results of transmission electron microscopy and scanning electron microscopy showed that the particles were spherical and homogeneous. Through gel retardation assay and MTT assay, we found that there were no obvious differences in binding capability of PEI to mc-DNA and plasmid DNA, as well as in cytotoxicity. The results of dynamic light scattering showed that the size of PEI/mc-DNA was about 68 nm, a slight larger than that of PEI/plasmid DNA. Furthermore, the tumor cells transfected with mc-GFP showed higher GFP expression level than that of conventional plasmid. The same results were achieved in the cells treated with tumor-suppressor gene pten, assayed by RT-PCR and Western blot. It indicates that the system of PEI/minicircle DNA is promising in gene transfer.

Effect of cimetidine on clonal expansion of TCR V? subfamily of T cells in cord blood stimulated by K562 cells / 第三军医大学学报

Objective To investigate the effect of cimetidine on the clonal expansion of TCR V? subfamily of T cells in cord blood after the stimulation by K562 cells in vitro.Methods Cimetidine(1?10-5 mol/L) or K562 cells(1?106/ml) or both of them were respectively cultured with mononuclear cells(MNC) isolated from normal human cord blood for 2 weeks.After the induction,specific cytotoxicity of the proliferated T cells were detected with K562 cells as the target cells.The selective usages and clonal expansion of TCR V? subfa-mily of T cells were analyzed by RT-PCR and genescan technique.Results After induction for 2 weeks,the 3 groups showed the increased cell proliferation,in which specific cytotoxicity of T cells induced by both cimetidine and K562 cells against K562 cells was enhanced significantly compared with the other 2 groups(P

Comparison of expansion of human ??T lymphocytes with solid phase antibody,HSP_(70BCG) and HSP_(70m)-peptide complexes in vitro / 中国免疫学杂志

Objective:To compare the human ??T lymphocytes expanded with HSP 70BCG and HSP 70 m-peptide complexes with those expanded with solid phase antibody in vitro.Methods:PBMCs were cultured with anti-TCR ?? antibody?HSP 70BCG and HSP 70 m -peptide complexes.The total cell number was counted.The flow cytometer was used to analyze the lymphocytes phenotypes and subtypes.RT-PCR was used to analyze the level of V? mRNA.The cytotoxitic activity of ??T cells against Daudi was determined using MTT calorimetric assay.Results:After 14 days of culture,in the antibody culture system,the total cell number increased about 30-40 fold,and the ratio of ??T cell reached to 86.3%;In the HSP 70BCG culture system,the total cell number increased about 7-8 fold,and the ratio of ??T cell reached to 71.23%;In the HSP 70-peptide complexes culture system,the total cell number increased about 4-5 fold,and the ratio of ??T cells reached to 27.26%.The antibody and HSP 70BCG activated ??T cells possessed a whole repertoire and mainly expressed V?9/V?2 subset and exhibited a strong cytotoxicity against Daudi.Conclusion:Anti-TCR ?? antibody and HSP 70BCG were able to proliferate purer ??T cells.The cells had a whole repertoire,and exhibited strong cytotoxicity against Daudi.