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Objetive: To investigate the long-term expression of the piggyback (PB) transposon system expressing human interleukin-6 (IL-6), nterleukin-3 (IL-3), interleukin-15 (IL-15), stem cell factor (SCF) and granulocyte-macrophage ctimulang Factor (GM-CSF) genes in the immunodeficient mice, and to provide a simple, long-term and new method for improving the reconstruction of human immune cells in the humanized mouse models. Methods: The PB transposon plasmid (PB-GFP) containing the GFP gene was constructed, and the PB transposon plasmid (PB-5F) contaning human IL-6, IL-3, IL-15, SCF, and GM-CSF genes was constructed. The 293T cells were divided into negative control group (non-transfection), postive control group (transfected with pLVTHM), transient transfection group transfected with PB-GFP plasmid and stable transfection group transfected with PB-GFP plasmid together with transposase plasmid (super-PB)]. The proportions of GFP cells in varions groups were measured by flow cytometry every three days after transfection. The NOD. Cg-Prkdcscid IL2rgun1wj1/SzJ (NCG) mice were divided into transient transfection group and stable transfection group. The mice in transient transfection group were transfected with PB-GFP alone, and the mice in stable transfection group were transfected with PB-5F and super-PB. On the 1st, 4th, 5th and 9th days and the followng every week after the transfection, the blood samples were collected, and the serum was separed; the levels of IL-6, IL-3, IL-15, SCF, and GM-CSF in serum of the mice in various groups were detected by ELISA Results: At 30 d after transfection of PB-GFP, the percentage of GFP1 cells of the mice in stable transfection group (4. 61% + 0.42%) was significantly higher than those in postive control group (0.58% +0.05%) and transient transfection group (0.86%+ 0.10%) (P<0.05). At 30 d after transfection of PB-5F plasmid, the levels of serum IL-6, IL-15 and GM-CSF of the NCG mice in stable transfection group were significantly higher than those in transient transfection group (P
ABSTRACT
Animal model is an animal material with human mimic performance established in biomedical scientific research. It can be used as experimental basis for studies of experimental hypothesis and clinical hypothesis. It can shorten the research time and observe the whole process of disease occurrence, development or prevention and treatment. Human biomedical research is largely limited by the biological complexity. In order to overcome this limitation, based on the immunosuppressive characteristics of a severely immunodeficient ( SCID) or recombinant activated gene ( Ragnul ) in mice, humanized mouse models of human diseases can be established and have been widely used to study the underlying principles of human immunobiology and complex pathological mechanisms of human diseases. This approach has become one of the important ways to promote the development of medical sciences, with practicality and foresight. In this paper, the application and research progress of humanized mouse models are reviewed.
ABSTRACT
Animal model is an animal material with human mimic performance established in biomedical scientific research. It can be used as experimental basis for studies of experimental hypothesis and clinical hypothesis. It can shorten the research time and observe the whole process of disease occurrence, development or prevention and treatment. Human biomedical research is largely limited by the biological complexity. In order to overcome this limitation, based on the immunosuppressive characteristics of a severely immunodeficient ( SCID) or recombinant activated gene ( Ragnul ) in mice, humanized mouse models of human diseases can be established and have been widely used to study the underlying principles of human immunobiology and complex pathological mechanisms of human diseases. This approach has become one of the important ways to promote the development of medical sciences, with practicality and foresight. In this paper, the application and research progress of humanized mouse models are reviewed.
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Hepatitis B virus ( HBV) threatens human's health seriously, immune disorder is the main pathogenesis.HBV cannot naturally infect mouse liver, thus the researchers tried to established HBV mouse models to imitate the immunological pathogenesis of HBV infection.This review summarize various methods to establish HBV mouse models, including HBV transgenic technics, HBV in vivo liver-target transfection technics and HBV naturally infected humanized mouse technics etc.Their advantages, disadvantages and contributions to immunological studies were also analyzed, and the development of this area was also prospected.
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Objective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model .Methods The Inducible expression system need to construct two plasmids:pLNHXO1O2-Alb-GLUC-FMN2A -rtTA and pLNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector pLNHX , Albumin promoter gene ( Alb) and rtTA gene or uPA gene and ZsGreen were obtained by PCR reaction and inserted into pLNHX .The Gaussia enzyme fluorescent element ( GLUC) was used to monitor rtTA expression in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA, and the ZsGreen for uPA expression monitoring in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen.The correct constructed plasmids were transfected into packaging cell line GP 2-293 to gain recombinant viral particles .NIH/3T3 cells were infected with these viral particles and selected with G 418.Gene expression in the surviving cells was confirmed by the PCR method .Results The recombinant retroviral vectors harbouring target genes were successfully cloned .The rtTA gene in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed, and uPA can be induced to express in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline (Dox) when the plasmid transfected into the HepG-Tet-on cell.The constructed recombinant two retroviral vectors were transfected into GP 2-293 packaging cells respectively to gain infectious viral particles .Then,NIH/3T3 cells were infected with these viral particles and single-cell clones which stably expressed the transgenes were successfully established .Conclusion This study primarily established uPA inducible expression system , it laid a foundation for the murine model of inducible liver damage , and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying .
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Epstein-Barr virus (EBV) is etiologically associated with a variety of diseases including lymphoproliferative diseases, lymphomas, carcinomas, and autoimmune diseases. Humans are the only natural host of EBV and limited species of new-world monkeys can be infected with the virus in experimental conditions. Small animal models of EBV infection, required for evaluation of novel therapies and vaccines for EBV-associated diseases, have not been available. Recently the development of severely immunodeficient mouse strains enabled production of humanized mice in which human immune system components are reconstituted and express their normal functions. Humanized mice can serve as infection models for human-specific viruses such as EBV that target cells of the immune system. This review summarizes recent studies by the author's group addressing reproduction of EBV infection and pathogenesis in humanized mice.
Subject(s)
Animals , Humans , Mice , Arthritis, Rheumatoid , Autoimmune Diseases , Epstein-Barr Virus Infections , Haplorhini , Herpesvirus 4, Human , Immune System , Lymphoma , Models, Animal , Reproduction , Vaccines , VirusesABSTRACT
PURPOSE: Many researchers have tried to develop animal models that mimic the human immune system, e.g. a humanized mouse model, to improve the engraftment of hematopoietic stem cells and develop human immune cells in an animal model. This study evaluated the feasibility of the cultured human umbilical cord blood (hUCB)-derived CD34(+) cells for cell expansion, in Rag2(-/-)gamma(c)(-/-) mice, and establish co-transplantation with human fetal thymus/liver tissue (Thy/Liv) under the kidney capsule. METHODS: Co-transplantation of hUCB-derived CD34(+) cells with Thy/Liv was performed. The hUCB-derived CD34(+) cells were prepared by freshly thawing (G1) and culturing for 7 days with two types of cytokine combinations (G2, G3). The CD45(+) cell populations were measured at 6, 8, 10 and 16 weeks in the peripheral blood. The splenocytes were cultured with mitogenic stimuli (PHA -L or IL-2) at 20 weeks post- transplantation, and the proliferation of human immune cells was evaluated. RESULTS: There were no significant differences in the human CD45(+) cell populations at 6, 8, 10 and 16 weeks post-transplantation between the groups. In the cultured splenocytes at 20 weeks post-transplant with PHA-L or IL-2, there was remarkable expansion of CD3(+) cells in the three groups. Although no CD19(+) cells were detected in the spleen, human Ig G was detected in the sera of these mice. CONCLUSION: The cultured and expanded hUCB-derived cells with cytokine combinations might be a feasible cell source in humanized mouse modeling. In addition, human immune cells can be reconstituted from the co-transplantation of Thy/Liv and cultured hUCB-derived CD34(+) cells.