Generation Of A Dual Inducible Membrane Tethering CreER For Cell Lineage Tracing

Backgrounds:Genetic lineage tracing is a powerful tool to unraveling the origin,fate and plasticity of cells.Lineage tracing is also indispensable to providing spatial,temporal,and kinetic resolution at single-cell level of the mechanisms that underlie tissue remodeling under physical and pathological conditions.The Cre-loxP recombination system,including the tamoxifen-inducible CreER-loxP,is the most widely used technology for in vivo stem cell and progenitor cell tracing.However,Numerous studies have demonstrated that the CreER-loxP system exhibits leakiness to an extent,which means the occurrence of spontaneous cell labeling in the absence of tamoxifen induction.Leakiness in CreER-loxP results in persistence of controversies in a couple important fields in development,for example,mechanisms of β cell homestasis in adult mice and the existence of cardiac stem cells.Objectives:The aim of this study was to construct a dual inducible membrane-localized CreER-loxP system,which could reduce the unintended nucleus translocation,and therefore,decrease the leakiness in classical CreER-loxP system.Methods:We generate a novel dual inducible CreER-loxP system with superior labeling characteristics.This two-component system consists of membrane localized CreER(mCreER:CD8α-FRB-CS-CreER)and TEV protease(mTEVp:CD8α-FKBP-TEVp),which are fusion proteins incorporated with the chemically induced dimerization machinery.Rapamycin and tamoxifen induce sequentially dimerization of FKBP and FRB,cleavage of CreER from the membrane and translocation into the nucleus.To measure the recombination efficiency and leakiness,Ad293 cells or Ad293CR cells(Ad293 cells stably carrying a Cre reporter cassette)were transfected with the plasmids and analyzed by flow cytometry and confocal microscopy.Cell viability assay,Cell cycle analysis,Fluorescence immunostaining,quantitative real-time RT-PCR,RNA Interference,Co-immunoprecipitation and Western blot analysis were carried out to determine the sub-celluar localization,interaction with HSP90 and cytotoxicity of mCreER.Lastly,lentiviral particles carrying an expression cassette of CreER,mCreER,or mTEVp driven by the RIP promoter were constructed.Lenti-RIP-CreER and Lenti-RIP-mCreER/Lenti-CMV-mTEVp lentiviral particles were used to verify RIP specificity and cytotoxicity.Results:Quantitative analysis of the classical CreER-loxP system showed a 51.6-79.7%labeling at 24-48 h post induction,while the uninduced labeling was as high as 24.1-58.3%,which was equivalent to a leakage of about 47.2-74.1%.Induction with rapamycin,tamoxifen,or both led to a labeling of 4.8-8.5%,7.3-18.0%,and 15.6-33.9%respectively in the dual inducible CreER-loxP system.The labeling leakiness in Ad293CR cells reduced dramatically from above 70%to under 5%.Fusion protein CreER-EGFP was used to traced the localization of CreER.The results showed that uninduced nucleus EGFP reached 45.1-79.5%,whereas mCreER-EGFP appeared predominantly in the plasma membrane,barely detectable in the nucleus in the absence of rapamycin and tamoxifen.The results demonstrated that Membrane localization of CreER reduces the level of nuclear translocation.The above results also indicated that there is a synergy between rapamycin and tamoxifen in the mCreER system.To investigate the underlying mechanism,HSP90 was knockdown by siRNA.The protein and transcript level of HSP90 reduced to about 17.3%and 10.1%respectively at 72 h post transfection.Decreased level of HSP90 led to increase of unintended cleavage of mCreER,and thus abolished largely the tightness of the dual inducible system.The results showed further that viability of Ad293 cells transfected with the CreER system decreased significantly after induction with tamoxifen for 24-48 h.On the contrary,the mCreER system did not affect cell proliferation,suggesting membrane localization of CreER could reduce cytotoxicity.Cell cycle analysis by flow cytometry showed that expression of CreER increased the percentage of cells in G1 stage,while decreased the percentage of cells in the G2/M stages.mCreER also affected cell cycle progression,but not as sever as CreER.Genome damage was assessed by Western blot and immunostaining of γ-H2AX.The results showed that CreER increased γ-H2AX ammount significantly,however,less DNA damage was induced with mCreER.The protein and transcript levels of p53 were determined by Western blot and qRT-PCR analysis,which showed a similar pattern with that of γ-H2AX.Ad293 cells transduction with Lenti-RIP-CreER led to 12.4-24.8%of unintended cell labeling at 24-48 h post induction.On the other hand,transduction with Lenti-RIP-mCreE/Lenti-RIP-mTEVp resulted to labeling of a few Ad293 cells at 48 h post induction,which was only 7%compared to that with Lenti-RIP-CreER.Low cytotoxicity of mCreER in MIN6 islet beta-cells preserved not only cell viability but also insulin content and secretion in response to high glucose concentration.Conclusions:Membrane localization of CreER prevents unintended entry into the nucleus.When grafted with the chemically induced dimerization machinery,cleavage of mCreER and subsequent nuclear translocation could be induced sequentially by rapamycin and tamoxifen.This system has an extremely low cell labeling leakage and cytotoxicity without compromising labeling efficiency.Although has not been tested in in vivo study,this novel dual inducible CreER system will be invaluable to cell lineage tracing and genome engineering in a more precisely temporal and spatial controllable manner.