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Establishment of Reversible Immortalized Murine Pancreatic β Cells and Biological Characteristics Study

Author: JiangJinXing
Tutor: QiZuo
School: Jinan University
Course: Immunology
Keywords: Tet-on advanced Lentiviral vectors SV40T antigen Islet β cells Transfection Biological characteristics
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 45
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Abstract

Objective can be regulated Tet-on advanced systems and SV40T gene construct p immortalized mouse pancreatic cell lines was observed after recovery characteristics of islet p cell biology, assess reversibility immortalized mouse pancreatic β cell line security and after recovery functionality, to address sources of islet cell transplantation to provide experimental basis. Methods Gateway recombinant DNA technology to construct Tet-on advanced systems regulate the expression of lentiviral vectors pLV.EX2d.P/puro-EF1 A gt; rtTA (M2) and pLV.EX3d.P/neo-TRE gt; SV40T gt; IRES / eGFP. Liposome transfection method lipotamin-2000, both the recombinant lentiviral vector were transfected into the packaging cell 293FT, to obtain high titer virus particles; then purified virus particles co-transfected cultured primary mouse islet cells transfection added after 24 hours doxycycline induced expression SV40T, puromycin was added after 48 hours and the antibiotic G418 in vitro amplification capability screened immortalized murine islet cells, a plurality of immortalized cells by ELISA based clone insulin secretion, RT-PCR detection of pancreatic islet cell-associated factor p screened with physiological function p reversibly immortalized mouse pancreatic cell lines. Select a Tet-on cell lines under the control of the 10th generation RIB cells, remove the DOX-induced silencing SV40T immortalization gene obtained after resuming RIB cells. By RT-PCR and immunofluorescence to detect response after SV40T and islet p cell-associated genes at the mRNA and protein level; karyotype analysis, soft agar colony formation assay, cell proliferation assay, and glucose-stimulated insulin release test monitoring reversibility immortalized islet β cells before and after DOX regulation of safety and functional changes. Results The recombinant lentiviral vector pLV.EX2d.P/puro-EF 1 A gt; rtTA (M2) and pLV.EX3d.P/neo-TRE gt; SV40T gt; IRES / eGFP by PCR and electrophoresis analysis showed successfully constructed and validated a recombinant vector correctness; packaging cells were transfected 293FT, 24 hours after transfection can be seen pLV.EX3d.P/neo-TRE gt; SV40T gt; IRES / eGFP vector 293FT with green fluorescence expression, transfection efficiency up 90%, 48 hours after the two viruses supernatant collected and concentrated by crystal violet staining titers can reach 107 and 108 respectively; the obtained virus particles while transfection of mouse primary islet cells, transfection DOX added 24 hours after induction of terminal differentiation state can be seen in islet cell proliferation phenomena 48 hours after transfection, G418 and puromycin selection, stable transfected cells continue to proliferate, and the remaining cell death; cell clone The insulin-positive ELISA test with 20, then mouse pancreatic β cell-related genes by RT-PCR, cell clones can be screened to an expression of Pdx, Nkx6.1, Pax6, Insulin islet β cell-associated genes, successfully established a reversible immortalized mouse pancreatic p cell line (RIB series). RIB tied with DOX induced vigorous proliferation, after removal of DOX RIB reply to slow down cell proliferation, completely stop the growth after 5 days; RT-PCR and cell immunofluorescence RIB tied DOX induced gene and protein SV40T have expressed that in the short term to remove DOX stop proliferation did not express SV40T, islet β cell-associated marker gene and protein expression in response to both front and rear; Reply RIB before and after were normal diploid karyotype analysis, no its flat cloning experiments tumorigenicity; RIB Department glucose-stimulated insulin secretion of insulin release test is weak, ability to enhance insulin secretion after recovery, but still there is a gap in primary islet cells, which is about the normal amount of insulin secretion 1/3-1/2. Conclusion Tet-on advanced system regulation recombinant lentiviral vector pLV.EX2d.P/puro-EF1 A gt; rtTA (M2) and PLV.EX3d.P/neo-TRE gt; SV40T gt; IRES / eGFP established successfully RIB system, its prolonged survival in vitro proliferation ability, and normal karyotype detection of pancreatic β cell-associated gene and protein expression continues, reply RIB after stopping the proliferation of cell lines did not express immortalization gene SV40T, and insulin secretion capacity enhancement, but still with the normal secretion of insulin a gap.

Establishment of Reversible Immortalized Murine Pancreatic β Cells and Biological Characteristics Study

Abstract

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