ABSTRACT
The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.
Subject(s)
Humans , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , DNA , Gene Editing/methods , Streptococcus pyogenes/metabolismABSTRACT
OBJECTIVE@#Trefoil factor 3 plays a pivot role in oncogenic transformation, growth and metastatic extension of solid tumours besides mucosal protection. We screened the best siRNA sequence targeting human TFF3 by the transient-transfection of the lentiviral mediated shRNA into thyroid carcinoma K1 cells which secrete TFF3 themselves.@*METHOD@#Four siRNA transcription template hairpin structure target potential sites in human TFF3 mRNA sequence(132,170,258 and 537 bp,seperately) were selected and synthesized,as well as one negative shRNA(shRNAC). After annealing in vitro, insert pLVX-shRNA-puro construct recombinant plasmid, then enzyme digestion and sequencing analysis. The lentiviral-shRNAs were transient-transfected into K1 cells. TFF3 mRNA and protein levels were test by real-time PCR and western blot respectively in K1 cells at 48h post transient-transfected.@*RESULT@#Genetic mutations in two sequences of shRNA1~2, so the follow-up study terminated. The TFF3 expression were obviously inhibited in K1 cells at 48 hours post transient-transfected of shRNA3 and shRNA4. TFF3 (258-276) showed the highest silencing efficiency (TFF3 mRNA reduced 60.67% and TFF3 protein reduced 56.44%, P < 0.01) when the transfection efficiency was 76.83%.@*CONCLUSION@#pLVX-shRNA-puro-TFF3 expression plamid were successfully constructed and the highest efficiency sequences were screened. All these laid a foundation for further study about the function of TFF3 gene.
Subject(s)
Humans , Cell Line, Tumor , Genetic Vectors , Lentivirus , Peptides , Genetics , Plasmids , RNA, Small Interfering , Genetics , Transfection , Trefoil Factor-2ABSTRACT
Objective To identify risk factors and preventive measures of nosoconial infections in patients with non-Hodgkin lymphoma (NHL). Methods Clinical data of 65 NHL patients admitted from January to December 2007 were retrospectively analyzed. Results According to WHO classification (2001), 58 patients were with B-cell lymphoma, 7 were with T-cell lymphorna. All patients received CHOP regimen as initial chemotherapy and 23 of them were with nosecomial infections. Logistic regression analysis demonstrated that age, length of stay, pathological type, bone marrow involvement, levels of serum lactate dehydrogenase (LDH), beta2-microglobulin and invasive treatment were identified as risk factors of nosocomial infections. Respiratory tract infections and infections with gram-negative microorganisms were the most popular. Conclusion High nosocomial infection rate is found in NHL patients, and control of risk factors may effectively prevent nosocomial infections in NHL patients.
ABSTRACT
Objective:To explore the expression of B7-1,B7-2 and B7-H1 on tumor-infiltrating dendritic cells(TIDC) and on splenic dendritic cells(SDC),and to investigate TIDC-mediated and SDC-mediated T-cell function after blocking B7-H1 expression in these dendritic cells.Methods: The TIDCs and SDCs were isolated from tumor-bearing mice using anti-mouse CD11c magnetic beads.The expression of B7-1,B7-2 and B7-H1 on TIDC and SDC was analyzed using flow cytometer.T cells were co-cultured with TIDCs or SDCs for the mixed lymphocyte reaction(MLR),and monoclonal antibodies to B7-H1 or the isotype control antibodies were added to the MLR cultures.T-cell proliferation was assessed using XTT method and the secretion of IL-10 was detected using ELISA.Results: B7-1 and B7-2 positive TIDCs were significantly less than SDCs(P0.05).T-cell proliferation stimulated by TIDCs was weaker than that stimulated by SDCs;T cells produced more IL-10 after TIDCs stimulation than after SDCs stimulation(P