Construction of Sox9 gene eukaryotic expression vector and its inductive effects on directed differentiation of bone marrow stromal cells into precartilaginous stem cells in rats

Abstract

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.

Keywords

precartilaginous stem cells / Sox9 / bone marrow-derived stromal cells / transfection

References

[1] RobinsonD., HasharoniA., CohenN., et al. . Fibroblast growth factor receptors-3 as a marker for precartilaginous stem cells. Clin Orthop, 1999, 357(Suppl): 163-175

[2] RobinsonD., AshH., YayonA., et al. . Characteristics of cartilage biopsies used for autologous chondrocytes transplantation. Cell Transplant, 2001, 10(2): 203-208

[3] CaplanA.I.. Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005, 11(7–8): 1198-1211

[4] ZhangS., ChenA., HuW., et al. . Immunological purification of rat precartilaginous stem cells and construction of the immortalized cell strain. Arch Orthop Trauma Surg, 2008, 128(11): 1339-1344

[5] KadomatsuH., MatsuyamaT., YoshimotoT., et al. . Injectable growth/differentiation factor-5-recombinant human collagen composite induces endochondral ossification via Sry-related HMG box 9 (Sox9) expression and angiogenesis in murine calvariae. J Periodontal Res, 2008, 43(5): 483-489

[6] ShibataS., Yokohama-TamakiT.. An in situ hybridization study of Runx2, Osterix and Sox9 in the anlagen of mouse mandibular condylar cartilage in the early stages of embryogenesis. J Anat, 2008, 213(3): 274-283

[7] HaagJ., GebhardP.M., AignerT.. SOX gene expression in human osteoarthritic cartilage. Pathobiology., 2008, 75(3): 195-199

[8] FriedensteinA.J., ChailakhyanR.K., GerasimovU.V.. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet, 1987, 20(3): 263-272

[9] KhanS.H., BrittoJ.A., EvansR.D., et al. . Expression of FGFR-2 and FGFR-3 in the normal human fetal orbit. Br J Ophthalmol, 2005, 89(12): 1643-1645

[10] TojyoI., YamagutiA., OzakiH., et al. . The expression of fibroblast growth factor receptor-3 in synovial osteochondromatosis of the temporomandibular joint. Arch Oral Biol., 2004, 49(7): 591-594

[11] XuK., GuoF., ZhangS., et al. . Blocking Ihh signaling pathway inhibits the proliferation and promotes the apoptosis of PSCs. J Huazhong Univ Sci Technolog [Med Sci], 2009, 29(1): 39-44

[12] PacificiM., KoyamaE., IwamotoM.. Mechanisms of synovial joint and articular cartilage formation: recent advances, but many lingering mysteries. Birth Defects Res C Embryo Today, 2005, 75(3): 237-248

[13] KinkelM.D., YagiR., McBurneyD.. Age-related expression patterns of Bag-1 and Bcl-2 in growth plate and articular chondrocytes. Anat Rec A Discov Mol Cell Evol Biol, 2004, 279(2): 720-728

[14] OkazakiK., IwamotoY.. Matrix gene regulation in cartilage, 2006, 16(7): 1213-1219

[15] KumarS., ChandaD., PonnazhaganS.. Therapeutic potential of genetically modified mesenchymal stem cells. Gene Ther, 2008, 15(10): 711-715

[16] FosterJ.W., Dominguez-SteglichM.A., GuioliS., et al. . Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY related gene. Nature, 1994, 372(6506): 525-530

[17] WagnerT., WirthJ., MeyerJ., et al. . Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene Sox9. Cell, 1994, 79(6): 1111-1120

[18] AkiyamaH.. Sox family regulate chondrogenesis. Clin Calcium, 2006, 16(2): 368-372

[19] IkedaT., KawaguchiH., KamekuraS., et al. . Distinct roles of Sox5, Sox6, and Sox9 in different stages of chondrogenic differentiation. J Bone Miner Metab, 2005, 23(5): 337-340

[20] PulkkinenH.J., TiituV., ValonenP., et al. . Recombinant human type II collagen as a material for cartilage tissue engineering. Int J Artif Organs, 2008, 31(11): 960-969

[21] GuimontP., GrondinF., DuboisC.M.. Sox9-dependent transcriptional regulation of the proprotein convertase furin. Am J Physiol Cell Physiol, 2007, 293(1): C172-C183

[22] MagdalouJ., NetterP., Fournel-GigleuxS., et al. . Agrecan and articular cartilage: assessment of glycosyltransferases for the restoration of cartilage matrix in osteoarthritis. J Soc Biol, 2008, 202(4): 281-288

[23] SekiyaI., VuoristoJ.T., LarsonB.L., et al. . In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA, 2002, 99(7): 4397-4402

[24] PanQ., YuY., ChenQ., et al. . Sox9, a key transcription factor of bone morphogenetic protein-2-induced chondrogenesis, is activated through BMP pathway and a CCAAT box in the proximal promoter. J Cell Physiol, 2008, 217(1): 228-241

[25] AbdallahB.M., KassemM.. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther, 2008, 15(2): 109-116

[26] KoyamaE., ShibukawaY., NagayamaM., et al. . A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis. Dev Biol, 2008, 316(1): 62-73

[27] AtkinsG.J., KostakisP., WelldonK.J., et al. . Human trabecular bone-derived osteoblasts support human osteoclast formation in vitro in a defined, serum-free medium. Cell Physiol, 2005, 203(3): 573-582

[28] van den DolderJ., JansenJ.A.. Enrichment of osteogenic cell populations from rat bone marrow stroma. Biomaterials., 2007, 28(2): 249-255

[29] KassemM., AbdallahB.M., SaeedH.. Osteoblastic cells: differentiation and trans-differentiation. Arch Biochem Biophys., 2008, 473(2): 183-187