Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add filters








Year range
1.
Protein & Cell ; (12): 31-42, 2019.
Article in English | WPRIM | ID: wpr-757943

ABSTRACT

Recently we have established a new culture condition enabling the derivation of extended pluripotent stem (EPS) cells, which, compared to conventional pluripotent stem cells, possess superior developmental potential and germline competence. However, it remains unclear whether this condition permits derivation of EPS cells from mouse strains that are refractory or non-permissive to pluripotent cell establishment. Here, we show that EPS cells can be robustly generated from non-permissive NOD-scid Il2rg mice through de novo derivation from blastocysts. Furthermore, these cells can also be efficiently generated by chemical reprogramming from embryonic NOD-scid Il2rg fibroblasts. NOD-scid Il2rg EPS cells can be expanded for more than 20 passages with genomic stability and can be genetically modified through gene targeting. Notably, these cells contribute to both embryonic and extraembryonic lineages in vivo. More importantly, they can produce chimeras and integrate into the E13.5 genital ridge. Our study demonstrates the feasibility of generating EPS cells from refractory mouse strains, which could potentially be a general strategy for deriving mouse pluripotent cells. The generation of NOD-scid Il2rg EPS cell lines permits sophisticated genetic modification in NOD-scid Il2rg mice, which may greatly advance the optimization of humanized mouse models for biomedical applications.

2.
Protein & Cell ; (12): 20-30, 2019.
Article in English | WPRIM | ID: wpr-757942

ABSTRACT

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.

3.
Protein & Cell ; (12): 154-155, 2019.
Article in English | WPRIM | ID: wpr-757929

ABSTRACT

In the original publication Fig. 1D and supplementary material is incorrect. The correct figure and supplementary material is provided in this correction.

4.
Chinese Journal of Biotechnology ; (12): 595-604, 2014.
Article in Chinese | WPRIM | ID: wpr-279480

ABSTRACT

Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.


Subject(s)
Blotting, Western , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Membrane Glycoproteins , Allergy and Immunology , Pichia , Metabolism , Plasmids , Recombinant Proteins , Single-Chain Antibodies
5.
Chinese Journal of Biotechnology ; (12): 1765-1775, 2013.
Article in English | WPRIM | ID: wpr-242455

ABSTRACT

Although DNA vaccination is now a promising strategy against hepatitis B virus (HBV) infection, this approach has relatively modest antiviral effect, indicating that immunosuppressive mechanisms may occur in the long-term established infection. In this study, we studied the immunogenicity and anti-HBV efficiency of a combination of HBV surface (HBsAg) and core (HBcAg) DNA vaccine, enhanced by heat shock protein (HSP) gp96 or HSP70 and mediated by in vivo electroporation. Immunization with gp96 adjuvanted HBsAg/HBcAg DNA formulation induced potent T cell and antibody immunity against HBsAg and HBcAg. Notably, treatment with gp96 or HSP70 as adjuvant resulted in reduction of Treg populations by around 20%. Moreover, compared with nonimmunized control mice, immunization with gp96 or HSP70 adjuvanted DNA vaccine dramatically decreased serum HBsAg and viral DNA levels, and HBcAg expression in liver. These results may therefore provide an effective strategy for designing gp96-based DNA vaccine for immunotherapy of chronic HBV infection.


Subject(s)
Adjuvants, Immunologic , Animals , Electroporation , HSP70 Heat-Shock Proteins , Allergy and Immunology , Hepatitis B Core Antigens , Allergy and Immunology , Hepatitis B Surface Antigens , Allergy and Immunology , Hepatitis B Vaccines , Allergy and Immunology , Hepatitis B, Chronic , Immunization , Membrane Glycoproteins , Allergy and Immunology , Mice , Mice, Transgenic , Vaccines, DNA , Allergy and Immunology
6.
Chinese Journal of Biotechnology ; (12): 261-266, 2012.
Article in Chinese | WPRIM | ID: wpr-304495

ABSTRACT

Heat-shock protein gp96 associates with antigenic peptides derived from tumor and virus. Exogenous gp96-peptide complexes are taken up by antigen-presenting cells through interaction with its receptor CD91 on the cell surface, and cross-present antigenic peptides to MHC class I molecules by a peptide relay line in the endoplasmic reticulum for specific T-cell activation. Meanwhile, gp96 has been shown to initiate innate immune responses through interaction with toll-like receptor 2 and toll-like receptor 4. Recent studies have shown a gp96-mediated immune balance between CTL and Tregs. With the further understanding of counteracting immunosuppressive mechanisms in gp96-induced cellular immune responses, and establishment of high level production of recombinant gp96 by the yeast, gp96 appears to be a promising candidate for designing effective therapeutic vaccines against tumor and infectious diseases.


Subject(s)
Animals , Communicable Diseases , Therapeutics , Heat-Shock Proteins , Allergy and Immunology , Therapeutic Uses , Humans , Immunotherapy, Active , Methods , Neoplasms , Therapeutics , T-Lymphocytes, Regulatory , Allergy and Immunology
7.
Protein & Cell ; (12): 364-371, 2012.
Article in English | WPRIM | ID: wpr-757263

ABSTRACT

As the most abundant liver-specific microRNA, microRNA-122 (miR-122) is involved in various physiological processes in hepatic function as well as in liver pathology. There is now compelling evidence that miR-122, as a regulator of gene networks and pathways in hepatocytes, plays a central role in diverse aspects of hepatic function and in the progress of liver diseases. This liver-enriched transcription factors-regulated miRNA promotes differentiation of hepatocytes and regulates lipid metabolism. With regard to liver diseases, miR-122 was shown to stimulate hepatitis C virus (HCV) replication through a unique and unusual interaction with two binding sites in the 5'-UTR of HCV genome to mediate the stability of the viral RNA, whereas inhibit the expression and replication of hepatitis B virus (HBV) by a miR-122-cylin G1/p53-HBV enhancer regulatory pathway. In addition, miR-122 acts as a suppressor of cell proliferation and malignant transformation of hepatocytes with remarkable tumor inhibition activity. Notably, a clinical trial targeting miR-122 with the anti-miR-122 oligonucleotides miravirsen, the first miRNA targeted drug, has been initiated for treatment of HCV infection. With further understanding of the comprehensive roles of miR-122 in hepatic functions and the mechanisms involved in miR-122 down-regulation in chronic hepatitis or hepatocellular carcinoma, miR-122 appears to be a promising candidate for effective therapeutic approaches against tumor and infectious diseases.


Subject(s)
Humans , Lipid Metabolism , Genetics , Liver , Metabolism , Liver Diseases , Genetics , MicroRNAs , Genetics , Metabolism , Models, Biological
SELECTION OF CITATIONS
SEARCH DETAIL