Construction Of IGF2Eukaryotic Expression Vector And Transgenic Research In Sheep Skeletal Muscle Cells

Insulin-like growth factor2(insulin-like growth factor2, IGF2), also known asgrowth regulators A (somatomedio A).It is a protein composed of67amino acid residues.Itplays an important role in regulating fetal development, tumor proliferation and musclegrowth.IGF2is a major growth factor in promoting mitosis of cells,but also is an autocrinesignal in the process of muscle cell growth.The proportion of skeletal muscle in variousorganizations of animals is40-60%.For livestock，the condition of growth and development ofmuscle tissue directly determines the meat and meat production. Recently, more and morestudies are about the IGF2function in growth and development of model animal.The studiesabout large mammal are few and the molecular mechanism of muscle cell growth anddevelopment regulated by IGF2is not understood clearly yet.The present study researched theregulatory role of IGF2on muscle cell proliferation and differentiation by transgenic methodsand the results were shown following:(1) The constructing of eukaryotic expression vector of IGF2The total RNA of sheep fibroblast is extracted by Trizol and made into cDNA by reversetranscription PCR.The primers were designed by IGF2mRNA sequence published inGenBank and IGF2gene is amplified successfully by RT-PCR.To construct the recombinantvector named pEGFP-N1-IGF2by series of processes such as digestion, ligation andtransformation.And the backbone vector is PEGFP-N1, the restriction sites is EcoR I/BamHI.The eukaryotic expression vectors is identified by methods of PCR, restriction digestion andsequencing and can be used to Transgenic Research.(2) Isolation and identification of the sheep skeletal muscle satellite cellsSheep skeletal muscle satellite cells are isolated successfully by the method of Two-stepenzymatic digestion.Morphological characteristics of freshly isolated muscle satellite cells isspherical, strong refraction.Adherent cells are spindle-shaped and begain to slowlyproliferation in2d.It start to rapid proliferation during3-5days and this be in the logarithmicphase.Differentiation medium induced cultured for4days and numbers of muscle cell fuse to form myotubes with skeletal muscle satellite cells typical biological characteristics can beobserved. Muscle satellite cells were identified in the molecular point of view byamplification of muscle-specific factors MyoD, myogenin.(3) The role of IGF2in skeletal muscle cell proliferation and differentiationUsing lipofectamine2000-mediated gene transfer method to optimize transfectionconditions of muscle cell, the recombinant vector pEGFP-N1-IGF2is transfed into sheepskeletal muscle cells and the transfection efficiency is35%,compared with other treatmentgroups, it is significant differences(P＜0.05).The result of semi-quantitative RT-PCR showsthat there is a upward trend of MyoD, myogenin expression in IGF2transfectedcells,compared with controlt group, it is significant differences(P＜0.05). Cell cycle analysisshowed that the percentage of S phase in transfected cells was significantly different(P＜0.05),compared with the control group.The stable transfected cell clones got by G418screening,IGF2transfected cell lines are established by the expanding culture, laid the basis for prepareIGF2clone animals by nuclear transfer.