Development of porcine induced pluripotent stem cells with a CD163 reporter system / 生物工程学报

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Construction of SH-SY5Y cell line stably expressing human α-secretase adam10 gene promoter luciferase reporter gene / 重庆医学

Objective To construct the luciferase report vector carrying a disintegrin and metalloprotease 10(adam10) gene promoter,to screen its stable expression cell line and to analyze its activity.Methods The genome DNA of human neuroblastoma SH-SY5Y cells was extracted as the template.The adam10 gene promoter was amplified by PCR and was cloned into luciferase reporter vector pGL4.17.The adam10 gene promoter luciferase reporter vector pGL4.17-adam10 was constructed and transfected in to SH-SY5Y cells(pGL4.17 vector without promotoer as the negative control and pGL4.17 vector with CMV promoter as the positive control).Then the stable expression cell line was screened by G418 and its fluorescence activity was detect after treating with 1 tμmol/L retinoic acid(RA) for 4 d.Results About 438 bp adam10 gene promoter was successfully amplified by PCR.The pGL4.17-adam10 vector was correct by pCR and double enzyme digestion identification.The cell line stably expressing adam10 gene promoter was obtained after transfecting SH-SY5Y cells by this vector and screening by G418,which had stronger transcriptional activity by detection;1 μmol/L RA could induce high efficiency expression of adam10 gene promoter.Conclusion Human adam10 gene promoter luciferase vector is successfully constructed.adam10 gene promoter can be stably expressed in SH-SY5Y cells,which provides a basis for deeply studying adam10 gene expression regulation,polymorphism analysis and high-throughput drug screening.

Construction of SH-SY5Y cell line stably expressing human α-secretase adam10 gene promoter luciferase reporter gene / 重庆医学

Objective To construct the luciferase report vector carrying a disintegrin and metalloprotease 10(adam10) gene promoter,to screen its stable expression cell line and to analyze its activity.Methods The genome DNA of human neuroblastoma SH-SY5Y cells was extracted as the template.The adam10 gene promoter was amplified by PCR and was cloned into luciferase reporter vector pGL4.17.The adam10 gene promoter luciferase reporter vector pGL4.17-adam10 was constructed and transfected in to SH-SY5Y cells(pGL4.17 vector without promotoer as the negative control and pGL4.17 vector with CMV promoter as the positive control).Then the stable expression cell line was screened by G418 and its fluorescence activity was detect after treating with 1 tμmol/L retinoic acid(RA) for 4 d.Results About 438 bp adam10 gene promoter was successfully amplified by PCR.The pGL4.17-adam10 vector was correct by pCR and double enzyme digestion identification.The cell line stably expressing adam10 gene promoter was obtained after transfecting SH-SY5Y cells by this vector and screening by G418,which had stronger transcriptional activity by detection;1 μmol/L RA could induce high efficiency expression of adam10 gene promoter.Conclusion Human adam10 gene promoter luciferase vector is successfully constructed.adam10 gene promoter can be stably expressed in SH-SY5Y cells,which provides a basis for deeply studying adam10 gene expression regulation,polymorphism analysis and high-throughput drug screening.

Construction and evaluation of the luciferase reporter vector from 3'-UTR of mice MIA3 gene / 中华显微外科杂志

Objective To construct MIA3 psicheck2 wild-type and mutant vectors targeting miR-374b,and provide the previous guarantee for the dual luciferase reporter assay.Methods The amplification primer was firstly designed according to mice MIA3-3'UTR sequence information,mice whole blood genomic DNA was taken as the template for PCR amplification of MIA3-3'UTR sequence,and the PCR product was cloned into psicheck2 dual luciferase reporter vector.Then,mutant primer was designed to mutate the MiR-374b seed sequence target TATTATA into AAATTAT so as to construct mutant vector.At last,the vector enzyme digestion evaluation and sequencing method was used to evaluate the constructed vectors.Results It could be seen from the analysis of agarose electrophoresis that the PCR amplification size of vector was consistent with the theoretical size.DNA sequencing evaluation showed that the MIA3-3'UTR-WT vector had been constructed successfully.The construction of mutant vector has successfully mutated the MiR-374b seed sequence target TATTATA into AAATTAT.Conclusion The successful construction of the vector will lay a foundation for the further evaluation on whether there is an actual binding site between the miR-374b and the chondrogenic differentiation-related target gene MIA3.

Construction of Iuciferase reporter vectors harboring DC-SIGN promoters without AP-1 or ETS-1 transcription factor binding site and detection of their activity / 中华微生物学和免疫学杂志

Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites (TFBS) in activity of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) pro-moter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/En-hancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% (293) and 10% (Hacat), which was 40%-50% with enhancer. The activity of DC-SIGN pro-moters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-1 TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter.

A New Reporter Vector System Based on Flow-Cytometry to Detect Promoter Activity

In this study, we report the development of a new dual reporter vector system for the analysis of promoter activity. This system employs green fluorescence emitting protein, EGFP, as a reporter, and uses red fluorescence emitting protein, DsRed, as a transfection control in a single vector. The expression of those two proteins can be readily detected via flow cytometry in a single analysis, with no need for any further manipulation after transfection. As this system allows for the simultaneous detection of both the control and reporter proteins in the same cells, only transfected cells which express the control protein, DsRed, can be subjected to promoter activity analysis, via the gating out of all un-transfected cells. This results in a dramatic increase in the promoter activity detection sensitivity. This novel reporter vector system should prove to be a simple and efficient method for the analysis of promoter activity.