The Basic Research Of Induced Pluripotent Stem Cells Using Recombinant Proteins

Part1Preparation of Recombinant PTD-Oct4 with Penetrating Peptide and Activity AssayObjective:Aim to establish the prokaryotic expression system of recombination PTD-Oct4-6His protein prokaryotic expression system of transcription factor gene with penetrating membrane activity. Methods:Using overlap extension PCR and prokaryotic expression vector PKYB, the Oct4 gene was expressed as a fusion protein PTD-KLF4 combined with PTD by gene splicing. The plasmid transfected to Ecoli ER2566.The fusion protein PTD-Oct4 was purified by Ni2+ affinity chromatography and identified with western blotting analysis. The fluorescein isothiocyanate (FITC) labeled PTD-Oct4-6His was used to investigate the penetrating ability of the fusion protein into Chinese hamster ovary (CHO) cells. And the bioactivity of PTD-Oct4 binding the target DNA sequence was detected by fluorescence resonance energy transfer (FRET). Result The results showed that the recombinant PTD-Oct4 successfully entered the cells and located in the nucleus with the transmembrane efficiency of 28.3%±2.4%. The recombinant PTD-Oct4 had specific binding capacity with its specific target DNA sequence. Conclusion The study founds basis for the preparation of the recombinant PTD-Oct4-6His and use it to induce target cells to become IPS. Key words Oct4; Induced pluripotent stem cells; FRET (Fluorescence resonance energy transfer)Part 2The culture of primary mouse corneal keratocytesObjective To improve the isolation and culture ways of mouse keratocytes, and to provide better seeding cells for the research of keratocyte biology, using mouse keratocytes as target cells for IPS induction and tissue engineering of cornea. Methods Primary mouse keratocytes were isolated and cultured by using Dispase digestion and culture of stroma tissue pieces. The growing characteristics of keratocyres were observed under inverted microscope. The cultured cells were differentiated by immunochemically staining with antibodies of Vimentin, and by RT-PCR for gene expression of Vimentin, Lumican and CK12. Result Mouse primary keratocytes showed triangle or dendritic morphology with many intercellular joints which could form networks. Confluences were reached on 6 day of culture. All of immunochemically staining of Vimentin were positive. RT-PCR indicated gene expressions of Vimentin and Lumican were positive and CK12 negative. Conclusion Keratocytes can be obtained by using Dispase digestion and culture of stroma tissue pieces. This research establishes a simple and highly efficient way for in vitro culture of pure mouse corneal keratocytes.Part3Recombinant Sox2,Oct4,Klf4,c-Myc with Penetrating Peptide and Activity Assay in mouse keratocytesObjective Aim to establish the eukaryotic expression system of recombinant nuclear factors Sox2,Oct4,Klf4,c-Myc protein with penetrating membrane and transfect to mouse corneal keratocytes. The protein expression will be evaluated. Methods Using overlap extension PCR and eukaryotic expression vector PCDNA3.1, the Sox2,Oct4,Klf4,c-Myc genes were expressed by gene splicing. The recombinant DNA sequencings were detected and evaluated whether or not the cloning was correct sequence. The plasmid transfected to mouse corneal keratocytes after right DNA sequencing was confirmed. The growing characteristics of keratocyres were observed under inverted microscope. The phenotype of transfected keratocytes were tested with Immunofluorescence staining of Sox2,Oct4,Klf4 for 10 days after transfection. Result The plasmids of PCDNA3.1-Sox2, PCDNA3.1-Oct4, PCDNA3.1-Klf4 and PCDNA3.1-c-Myc were successfully constructed. The recombinant DNA sequencings were correct. The plasmids successfully transfected to mouse corneal keratocytes and caused cell shape changes. The transfected keratocytes were positively stained with Sox2,Oct4,Klf4 for 10 days after transfection. Conclusion The eukaryotic expression system of recombinant nuclear factors Sox2,Oct4,Klf4,c-Myc protein with penetrating membrane is successfully established, which can transfect to mouse corneal keratocytes, and cause cell shape and phenotype changes. The study founds basis for eukaryotic recombinant nuclear factors to induce keratocytes to become IPS.