Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立

doi:10.16431/j.cnki.1671-7236.2016.01.003

Abstract

Abstract: Sox2 is one important marker of pluripotent stem cells, a study found that neural stem cells with high expression of Sox2 as donor cells showed higher reprogramming ability in nuclear transplantation.In this study, through enhancing exogenous Sox 2 gene expression of sheep bone marrow mesenchymal stem cells, in order to raise their reprogramming ability, and improve the efficiency of somatic cell cloning in animal.Total RNA was extracted from sheep testicular tissue, and with this template, Sox2 cDNA sequence was amplified and inserted into the eukaryotic expression vector pEGFP-N1 to build a recombinant vector pEGFP-N1-Sox2.The vector was transfected into the sheep bone marrow mesenchymal stem cells by liposome method, and through G418 and fluorescence screening to obtain and amplify monoclone.DNA sequencing showed that sheep Sox 2 gene CDS sequence was obtained, and recombinant plasmid was successfully constructed.Identification of fluorescence confirmed that stable sheep bone marrow mesenchymal stem cell lines transfected with Sox2 were established.This study obtained the sheep bone marrow mesenchymal stem cell lines with high expression of Sox2, and provided a new idea for raising reprogramming efficiency in the process of somatic cell cloning.

Key words: Sox2 gene; bone marrow mesenchymal stem cells; reprogramming

CLC Number:

Q813

LIU Huai-yu, LI Hao-tian, SU Xiao-hu, MENG Fan-hua, LIU Chun-xia, ZHANG Yan-ru, CAO Jun-wei. Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene[J]. , 2016, 43(1): 16-22.

References

[1] Kato Y,Imabayashi H,Mori T,et al.Nuclear transfer of adult bone marrow mesenchymal stem cells:Developmental totipotency of tissue-specific stem cells from an adult mammal[J]. Biology of Reproduction,2004,70(2):415-418.
[2] 张焱如,刘宗正,韦林盖,等.蒙古马骨髓间充质干细胞的分离培养及多向分化潜能的研究[J].畜牧兽医学报,2011,42(10):1357-1361.
[3] 韦林盖,周欢敏,张焱如,等.蒙古羊骨髓间充质干细胞的分离培养及多向诱导分化[J].中国畜牧兽医,2013,40(8):7-12.
[4] Kumar B M,Jin H F,Kim J G,et al.Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells[J]. Developmental Dynamics,2007,236(2):435-446.
[5] Mizutani E,Ohta H,Kishigami S,et al.Developmental ability of cloned embryos from neural stem cells[J]. Reproduction,2006,132(6):849-857.
[6] Blelloch R,Wang Z,Meisser A,et al.Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus[J]. Stem Cells,2006,24(9):2007-2013.
[7] Bosch P,Pratt S L,Stice S L.Isolation characterization gene modification and nuclear reprogramming of porcine mesenchymal stem cells[J]. Biology of Reproduction,2006,74(1):46-57.
[8] Inoue M,Kamachi Y,Matsunami H,et al.Pax6 and Sox2-dependent regulation of the Sox2 enhancer N-3 involved in embryonic visual system development[J]. Genes to Cells,2007,12(9):1049-1061.
[9] Avilion A A,Nicolis S K,Pevny L H,et al.Multipotent cell lineages in early mouse development depend on Sox2 function[J]. Genes & Development,2003,17(1):126-140.
[10] Rideout W M,Eggan K,Jaenisch R,et al.Nuclear cloning and epigenetic reprogramming of the genome[J]. Science, 2001,293(5532):1093-1098.
[11] Zappone M V,Galli R,Catena1 R,et al.Sox2 regulatory sequences direct expression of a b-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo revealing regionalization of gene expression in CNS stem cells[J]. Development,2000,127(11):2367-2382.
[12] Inoue K,Noda S,Ogonuki N,et al.Differential developmental ability of embryos cloned from tissue-specific stem cells[J]. Stem Cells,2007,25(5):1279-1285.
[13] Jin H F,Kumar B M,Kim J G,et al.Enhanced development of porcine embryos cloned frombone marrow mesenchymal stem cells[J]. Int J Dev Biol,2007,51(1):85-90.
[14] Takahashi K,Yamanaka S.Induction of pluripotent stemcells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell,2006,126(4):663-676.
[15] Lengerke C,Fehm T,Kurth R,et al.Expression of the embryonic stem cell marker Sox2 in early-stage breast carcinoma[J]. BMC Cancer, 2011,11:42.
[16] Liu X F,Yang W T,Xu R,et al.Cervical cancer cells with positive Sox2 expression exhibit the properties of cancer stem cells[J]. PLoS One,2014,9(1):e87092.
[17] Eini R,Stoop H,Gillis A J M,et al.Role of Sox2 in the etiology of embryonal carcinoma,based on analysis of the NCCIT and NT2 cell lines[J]. PLoS One,2014,9(1):e83585.
[18] Keramari M,Razavi J,Ingman K A,et al.Sox2 is essential for formation of trophectoderm in the preimplantation embryo[J]. PLoS One,2010,5(11):e13952.

Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene

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