Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

Complement and membrane-bound complement regulatory proteins as biomarkers and therapeutic targets for autoimmune inflammatory disorders, RA and SLE.

Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA.

Research on protein expression of CD55 in triple-negative breast cancer and its effect on prognosis / 中国医学装备

Objective:To investigate the expression of CD55 in triple-negative breast cancer (TNBC) and its correlations with prognosis.Methods: The expressions of CD55 was detected by immunohistochemistry Streptavidin-Peroxidase method in 100 cases of triple-negative breast cancer and 50 cases of adjacent-carcinoma tissues, with clinical and pathological features and follow-up data, the Cox model analysis was established and to analyze the prognostic factors. Results: The positive expression of CD55 was 56.0% in the tissues from triple-negative breast cancer, whereas its positive rate was 8.0% in the corresponding adjacent breast tissues, with statistical significance (x2=32.0,P<0.05). The expression of CD55 was significantly related with tumor size, clinical stage and axillary lymph node metastases (x2=7.404,x2=8.688,x2=4.802;P<0.05); the expression of CD55 did not show correlation to age, and histologic grade. Multivariate analysis showed that the age, clinical stage, CD55 protein expression and axillary lymph node metastasis were the independent risk factors affecting the 5-year disease-free rate of patients and that clinical stage and axillary lymph node metastasis were the independent risk factors affecting the 5-year overall rate of patients.Conclusion: The expression of CD55 might be related to the development and prognosis of triple-negative breast cancer. The expression of CD55 may be used as one of the indicators in determining the progress and prognosis of TNBC.

The Stromal Overexpression of Decay Accelerating Factor (DAF/CD55) Correlates with Poor Clinical Outcome in Colorectal Cancer Patients

BACKGROUND: Decay accelerating factor (DAF/CD55), regulates the complement system by accelerating decay of the C3 convertase, has been described in several malignancies, however, the clinicopathologic significance of CD55 and its receptor CD97 has not been fully investigated. We examined the expression patterns of both CD55 and CD97 and their association with clinicopathologic parameters in colorectal cancers (CRCs). METHODS: Expression patterns of CD55 and CD97 in the stroma and tumor cells at tumor center and invasive front were examined in 130 CRCs, and their significance was statistically evaluated. RESULTS: CD55-high stroma was correlated with tumor border (p=0.006) and invasion depth (p=0.013). CD55-high tumor cells at tumor center and invasive front were correlated with histologic grade, and CD55-high tumor cells at invasive front with tumor, node and metastasis (TNM) stage (p<0.05). CD97-high stroma was correlated with lymph node metastasis (p=0.016) and TNM stage (p=0.030). CD97-high tumor cells at tumor center and invasive front were correlated with tumor size and CD97-high tumor cells at tumor center with tumor border (p<0.05). Patients with CD55-high stroma showed poor overall and recurrence-free survival (p<0.05) in univariate analysis, and were independently associated with short recurrence-free survival (p=0.025) in multivariate analysis. CONCLUSIONS: Stromal CD55 overexpression would be an indicator of adverse clinical outcome and a useful prognostic factor.

Construction the eukaryotic expression vector of human decay accelerating factor and transfection NIH/3T3 cells / 医学研究生学报

Objective: To construct eukaryotic expression vector pSecTag2/HygroB-DAF of human decay accelerating factor (DAF) and transfect NIH/3T3 cells after encapsulated by chitosan. Methods:The human DAF fragments were obtained by PCR form DAF-pGEM-T Easy Vector, cloned into the eukaryotic expression vector pSecTag2/HygroB, and identified by restriction endonuclease’s digestion and DNA sequencing. After the particles of pSecTag2/HygroB-DAF were encapsulated by chitosan, the NIH/3T3 cells were transfected by chitosan-DAF nanoparticles and detected DAF expression by immunohistochemistry stain. Results:The DAF fragment was 1049 bp. Its sequence was as same as DAF cDNA in Genebank. After having been transfected by chitosan-DAF nanoparticles 24 hours, the NIH/3T3 cells showed diffusely positive in cytoplasms by anti-DAF immunohistochemistry. Conclusion:Eukaryotic expression vector of human DAF were constructed successfully and transfected it to NIH/3T3 cells after encapsulated by chitosan.

Expression of human hDAF in CHO cells and its decay-accelerating activity / 第三军医大学学报

Objective To obtain Chinese hamsterovary (CHO) cell line expressing human decay accelerating activity (hDAF) stably and to observe the protective effect of hDAF on heterologous cells under the circumstance of complement activation. Methods The eukaryotic expression vector DAF pcDNA3.1 was constructed and then transfected into CHO cells by lipofection. Monoclones of cells expressing hDAF stably were screened by the method of limiting dilution. hDAF expression was detected by flow cytometry. The decay accelerating activity of hDAF was determined by assay of C3 deposition and 51Cr release. Results The expression vector DAF pcDNA3.1 was successfully constructed, and monoclones of cells expressing hDAF were obtained. CHO cells expressing hDAF could decrease C3 deposition and attenuate the killing effect of activation of the complement system. Conclusion We have obtained CHO cell clones expressing hDAF stably, which is helpful for the further studies of the relationship of the structure with the functions of hDAF.

Expression of Complement Regulator Genes in Abeta1-42 Stimulated Human Neuroblastoma Cell

BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.

Expression of Complement Regulator Genes in Abeta1-42 Stimulated Human Neuroblastoma Cell

BACKGROUND: Endogenous complement inhibitors in the brain may protect against the neuroinflammation in Alzheimer's disease. Human neuroblastoma cells were stimulated by Abeta1 - 4 2 to investigate whether the expression of various complement regulator genes is upregulated. METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 microM of Abeta1-42 or 0.5 ng/ml - 5 ng/ml of TNFalpha or both. Actinomycin D (2.5 microM) or cycloheximide (2.5 microM) was also added to the cell suspension. Messenger RNA expression of decay accelerating factor (DAF), membrane cofactor protein (MCP), CD59, complement-receptor 1(CR1), C1 inhibitor (C1-INH), C4-binding protein, factor H, factor I, clusterin and S-protein was measured by RT-PCR. RESULTS: Abeta1-42 and TNFalpha upregulated the expression of C1- INH significantly but increased expression of mRNA for factor H was not statistically significant. The expression of mRNAs for DAF and MCP was at low a level after stimulation. Factor I, CD59 and clusterin were not changed in their mRNA level. The mRNAs for S-protein, C4-binding protein and CR1 were not detected. Actinomycin D suppressed mRNA levels of C1-INH and CD59 significantly. Cycloheximide also inhibited the expression of both C1-INH and CD59. CONCLUSIONS: Early upregulated expression of C1-INH in Abeta1-42 stimulated neuroblastoma cell may contribute to a host defense mechanism against complement-mediated neuronal cell damage.

Prediction of the secondary structure and B cell epitope of human decay-accelerating factor / 中国免疫学杂志

Objective:To predict the secondary structure and B cell epitope of human decay accelerating factor Methods:The accuracy of two secondary structure prediction methods,Chou & Fasman and PHDsec,was compared,and then human DAF secondary structure was analyzed by PHDsec Hydrophilicity scores and solvent accessibility of DAF SCR1 4 were obtained by methods of Hopp & Woods and PHDacc respectively Combined with the secondary structure of DAF,the B cell epitopes in DAF were predicted Results:PHDsec which use multiple sequence alignment,achieve a better prediction accuracy There is no alpha helix in SCR1 4 of human DAF but only beta sheet and loop segments; The computer predicted most possible epitope in DAF localize within amino acid residues Pro73 Val79?Arg130 Leu139 and Glu156 Cys163 Conclusion:These results will be useful in estimate of the epitope and activity sites localized within human DAF