Construction And Expression Of Key Transcription Factors Involved In Hematopoiesis In Lentivirus

The hematopoietic system have been studied extensively, for the therapeuticpurposes in the treatment of blood diseases. Hematopoiesis is a term used to describethe process of all types blood cell formation during both the embryonic and adult stagesof an organism. HSCs continuously differentiate into multiple lineages of differentblood cell types, simultaneously replicating themselves through self-renewal to preventdepletion of the stem cell pool in the bone marrow. With the studies of transgenic micemodel and the blood sample from people with leukemia，lots of transcription factorswere identified. Most of them involved in hematopoiesis and play important roles inboth self-renewal and lineage differentation.Most previous studies carried out on stem cell differentation focus on changing theextracelluar microenvironment. For instance,some kinds of cytokines were added toinduced a certain specify type of cell production. With the emerging of inducedpluripotent stem cells(iPS), regulation of the gene that involved in cell fate decisiondirecitly has been a new way to study cell differentation. Changing cell’s fate bytranscription factors is a new approach. The study of hematopoietic transcriptionfactor will help us understanding the mechanism of HSC differentation andself-renewal better.Lentivirus have the ability to infect almost all kinds of mammalian cells includingnon-divide cells. Using the lentivirus which have been modified,we can intergrate theexogenous gene into a host genome efficiently. In our study we cloned24transcriptionfactors that involved in hematopoiesis into lentivirus vector. They are：Bmi-1, C/EBP- α, C/EBP-β, C/EBP-ε, E2A, Ebf-1, Fli-1, Gata-1, Gata-2, Gata-3, Gfi-1, Ikaros,Lhx2, Lmo2, Msi-2, Oct-4, Pax-5, PU.1, Runx-1, Sall4, Scl/Tal, Sox17, Tel/Etv6andZfx. As far as we know Bmi-1，Gfi-1，CEBP-αplay enssential roles in HSCself-renewal,while some of others such as Runx-1,Lmo2,Tel/ETV6,Scl-Tal-1andGata-2involved in both HSC self-renewal and lineage differentation. These factorswere selected based on the literature of transgnic mice model or leukemia assay. We testtheir protein expression by virus packaging. Althogh HSC from bone marrow or cordblood have been succeed used in clinical triat. They can not been cultured in vitro for alonger time. We will make use of the transcription factors to induce fibroblasttransdifferentation into blood cells or hematopoietic stem cells. Further more we canexplore the key factors that sustain HSC self-renewal in vitro. This study will provideus a guide towards clinical application.Our work to construct and expression of the transcription factors in lentivirus willlay a solid foundation for the further study. Gene-targeting based on the embryonic stem cell technology and homologousrecombination,is used in alter the genome of a organism at a specific point. Generally ithas two approaches:gene knock-in and gene konck-out. The Cre/LoxP system mediatetissue-sepecific gene knock-out give us a new method to study of the gene which islethal to the embryonic when it was knockout.This system needs two kinds of genetic engineered mice. The Cre mice and thefloxed mice. And the construction of tissue-specific Cre targeting vector is a key pointin this project.The endothelial protein C receptor(EPCR) encoded by Procr gene,was specificexpressed in endotheliocyte. EPCR was involevd in protein C anticoagulant system,andwas also has a anti-inflammatory effect. And Procr has been detected in HSC andmonocytes.So far many kinds of tisuue-specific of cell-specific Cre mice have been widelyused in many studies.Such as the adipose tissue-specific Ap2-Cre mice, the nervousand the kidney podocyte specific Nestin-Cre mice,the B cell specific CD19-Cremice,the T cell specific LCK-Cre mice. But the endotheliocyte specific Cre was notwell studied. In this study,we use Procr as a endotheliocyte tissue specific promoter,andgenerate the Procr-CreERT2knock-in mice targeting vector by homologousrecombination. And the hematopoietic stem cell of the knock-in mice would be easilysorted out. The mice can also be widely used as a tool mice which can conduct a specifygene konck-out in endothelial cells.