Construction Of Tissue Specific Promoter By Gateway Technology And Application In Differentiation Studies Of Induced Pluripotent Stem Cells

ES cells are pluripotent cells derived from morula or the inner cell mass ofblastocyst-stage embryos. The abilities of ES cells to undergo indefiniteself-renewal in vitro and to produce derivative lineages of all three embryonicgerm layers in vitro and in vivo make them a highly prized reagent in both clinicaland research settings. Thus, embryonic stem cell become a hot spot inbio-engineering research and plays an important role in cytobiology,developmental biology, genetics, neurobiology and clinical medicine.Successful reprogramming of human somatic cells to pluripotent stem cells byectopic expression of defined transcriptional factors (Oct4, and Sox2with eitherKLF4and c-Myc or Nanog and Lin28) has been reported in the end of2007[4-8].It has been proved that iPS cells resemble ES cells in many aspects, including theexpression of certain stem cell markers, embryoid body formation, teratomaformation， chromatin methylation patterns, and viable chimera formation.Therefore, patient-specific iPS cells that established in culture will provide aninvaluable tool for study of developmental biology and drug discovery andunlimited cell resources for regenerative medicine without ethical conflicts. At present, numerous studies demonstrated that embryonic stem cells canproduce derivative lineages of all three embryonic germ layers. However, someproblems need to be solved before embryonic stem cell can be used for clinicalresearch. One is hard to purify target cells from the mixture differentiated cellpopulation. Two possible solutions for this problem mentioned above are to usecell-specific antibodies to mark target cells and use cell specific promoter drivingreporter genes system, so that we can collect the target cells through FACS.Moreover, the latter one has more advantages that when the target cells expressedreporter gene were transplanted into the body, we can easily observe andevaluation the cell delivery, differentiation, safety, toxicity, and efficacy.The reporter genes (such as fluorescent proteins) have become extremelyversatile tools for tracking and quantifying biological entities in the fields ofbiochemistry, molecular and cell biology, as well as high throughput screening andgene discovery. Reporter genes transferred into stem cells can be detected as atracer in in-vivo research and enable us to inspect the morphology change andmovement of stem cells in in-vitro research. In view of the important roles of EScells and transgenic techniques, the highly efficient transduction and persistentstable expression of exogenous genes, with little influence on biologicalcharacteristics, are of great necessity. Though there are different transgenicmethods, it is difficult to get a purified cell population by exogenous geneinfection due to the colony-like growth state, the complex growth environment andharsh culture condition. Until now there are different methods for transferringexogenous genes into ES cells, such as lipofection [11], electroporation [10],virus-mediated transduction including simian immunodeficiency virus (SIV)[15]and lentivirus. Among them lentivirus mediated transduction is an ideal method. Inorthodox plasmid construction, the complex procedure and possibility offalse-positive hinder the experiments. Recently, a nimble and universal clonemethod named Gateway technology emerged. Bacteriophage lambda att siterecombination is a well-characterized phenomenon. In bacteria, there is a stretch ofDNA called attB,(B stands for bacteria), and in the phage there is a stretch of DNA called attP (P stands for phage). When the phage infects a bacterium, theinjected lambda DNA recombines with the corresponding bacterial DNA via the attsites in the presence of integration-specific enzymes. When an attB siterecombines with an attP site, the outcome is integration of the phage DNA into thebacterial genome. Once integrated, the hybrid recombination sites are called attLand attR (L stands for left, R stands for right). These recombination reactions(“LR” and “BP”) are the basis of the Gateway Cloning System. Gateway cloning technology usually gets higher cloning efficiency than traditionalrestriction enzyme cloning and can simplify the cloning workflow and save time.As mentioned above, the purpose of this research is first to constructtissue-specific promoter driving fluorescent reporter system includingpLV/Final-puro-αSMA-hrGFP and pLV/Final-αMHC-hrGFP. Then iPSc weretransduced with pLV/Final-puro-αSMA-hrGFP. Purified transduced cells thatattained by antibiotic selection were induced to differentiate to α-smooth musclecells. Fluorescent cells emerged some days after induction and were identificatedand analized by immunofluorescence.Part1: Using Gateway technique to build up two lentivirus vectorsnamed pLVpuro/αSMA-hrGFP and pLVpuro/αMHC-hrGFP.Part2: Induced pluripotent stem cells were transduced withpLVpuro/αSMA-hrGFP containing reporter gene under the control of tissuespecific promoter and were induced to differentiate to specific cell lineages.Part one Construct of lentivectors by multisite gateway technology1.1ObjectiveIn this study, pLVpuro/αSMA-hrGFP and pLVpuro/αMHC-hrGFPlentiviral vectors were constructed by multisite gateway technology. ThenpLVpuro/αSMA-hrGFP and pLVpuro/αMHC-hrGFP plasmid vectorswere detected and biological safety was evaluated, for the purpose of constructing safe and effective lentivirus vectors.1.2Methods1.2.1LR clonase was applied to link two entry clones, pDONRTM221-reportergene, and pDONRTML4-promoter-R1and destination vector, to constructexpression vectors2K7puro/promoter-reporter gene.1.2.2C2C12cells and mouse cardiac myocytes were individually infected bylineated pLVpuro/αSMA-hrGFP and pLVpuro/αMHC-hrGFP lentivectors.48h to72h post-transfection, green fluorescence were seen under fluorescencemicroscopy.1.3Results1.3.1pLVpuro/αSMA-hrGFP and pLVpuro/αMHC-hrGFP lentivectors wereobtained after LR reaction and monoclonal bacteria were gained aftertransformation.1.3.248h to72h post-transfection, green fluorescence were seen underfluorescence microscopy in C2C12and mouse cardiac myocytes.1.4Conclusion1.4.1pLVpuro/αSMA-hrGFP and pLVpuro/αMHC-hrGFP lentivectorslentivectors were successfully constructed.1.4.2Transduced cells expressed GFP successfully. These confirmed thesuccessful construction of the aboved lentivectors lentivectors, and provide apowerful tool for the research of stem cells differentiation and in vivotransplantation studies. Part two: iPSCs were transduced with lentivirus containingreporter gene under the control of tissue specific promoter andwere induced to differentiate to specific cell lineages2.1ObjectiveIn this study, iPSCs cells were transduced by pLVpuro/αSMA-hrGFPlentivectors. Puromycin was added to the culture for selection and then puretransduced iPSCs cells population was attained. Different inducing factors wereintroduced to the culture to induce infected iPSCs cells differentiate to vescularsmooth cells. Fluorescent differentiated cells appeared5-25days after inductionand were analyzed by immunofluorescence.2.2Methods2.2.1Immunofluorescence was carried out for detail characterization ofFSK-iPSCs.2.2.2pLVpuro/αSMA-hrGFP lentivectors were transduced into FSK-iPSCs.7days after transduction,1-5μg/ml puromycin was added to the culture andselection was lasted for14days.2.2.3The transduced FSK-iPSCs were induced to differentiate for5days in vitro.The CD4+cells were obtained by fluorescence-activated cell sorting, and theninduced into vascular smooth cells in vitro. Differentiated cells were observedunder fluorescent microscope everyday after induction.2.3Results2.3.1FSK-iPSCs identification: The results of fluorescence immunocytocheminstryshowed FSK-iPSCs strongly expressed OCT-4and SSEA-1. Three germ layerstissues and cells was founded expressed during the nature differentiation process of FSK-iPSCs by fluorescence immunocytocheminstry.2.3.2FSK-iPSCs were successfully transduced by lentivirus. Through antibioticselection, pure infected cell population was attained, and the growth of cells wasnot influenced after virus infection.2.3.3The CD4+cells were obtained by fluorescence-activated cell sorting from thetransduced FSK-iPSCs.2.3.4The CD4+cells were then differentiated into vascular smooth cells in vitro.5-25days after differentiation, GFP-expressed cells appeared in the culture,immunofluorescence demonstrated that these fluorescent cells frompLVpuro/αSMA-hrGFP transduced FSK-iPSCs were indeed αSMA positive cells.2.4Conclusions2.4.1FSK-iPSCs have similar characteristics of mouse ES cells.2.4.2FSK-iPSCs were successfully transduced by pLVpuro/αSMA-hrGFPlentivirus. Through antibiotic selection, pure infected cell population was attained,transduced FSK-iPSCs differentiated to vascular smooth cells expressing αSMAsuccessfully. This reporter-gene containing vectors can reflect the expression ofαSMA. These cells provide a powerful tool for the research of stem cellsdifferentiation and in vivo transplantation studies.