Construction of Nanog lentiviral expression vector and its application in differentiation regulation of embryonic stem cells

doi:10.3969/j.issn.2095-4344.2016.19.004

Abstract

Abstract:

BACKGROUND: There is a lack of safe and effective modular lentivectors in differentiation regulation of embryonic stem cells.
OBJECTIVE: To construct a modular lentivector, Puro-Nanog-hrGFP, with Nanog promoter-controlled humanized renilla reniformis green fluorescent protein (hrGFP) and puromycin and to observe the expression of Nanog during the differentiation of Nanog-hrGFP-transfected mouse embryonic stem cell lines.
METHODS: After PCR amplification, Nanog, hrGFP and entry vector were recombined into the pDest-puro vectors to generate the lentiviral expression vector, Puro-Nanog-hrGFP, by the LR reaction. Through lentivirus production, transduction and puromycin screening, the transduced cell lines with Nanog-hrGFP gene were generated and identified. Expression of Nanog during the differentiation of transgenic mouse cell lines was detected.
RESULTS AND CONCLUSION: The lentiviral expression vectors Puro-Nanog-hrGFP were constructed successfully by Gateway technology, and then the transducted cell lines were obtained by lentiviral infection. The expression of Nanog was gradually decreased during the process of transgenic cell lines differentiation, which provides a new tool for further investigation on regulation of stem cell differentiation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Embryonic Stem Cells, Transgenes, Tissue Engineering

CLC Number:

R394.2

Chen Fei, Wan Li, Li Yan, Li Xin. Construction of Nanog lentiviral expression vector and its application in differentiation regulation of embryonic stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2763-2769.

Figures/Tables 2

2.1 入门克隆pup-Nanog的鉴定通过kana正选，氯霉素负选出来的克隆，进行质粒和PCR扩增，琼脂糖凝胶电泳显示PCR条带500 bp左右(如图3)，与Nanog大小相符，表明入门克隆pup-Nanog构建成功。 2.2 目的载体(Nanog-hrGFP)的鉴定慢病毒表达载体质粒(Nanog-hrGFP)PCR扩增目的载体中Nanog和hrGFP序列，琼脂糖电泳显示，Marker为1 kb，PCR鉴定结果与Nanog(500 bp左右)和hrGFP(720 bp)，结果发现PCR电泳图大小符合，表明慢病毒表达载体质粒(Nanog-hrGFP)构建成功(图4)。 2.3 慢病毒的包装将Nanog-hrGFP质粒与Vira PowerTM Lentiviral Packaging Mix共转染239FT细胞，约48 h后，镜下可见部分细胞融合，出现多核复合体；60 h后细胞融合和多核复合体增多，如图5，72 h收集病毒上清。超速离心浓缩病毒，并进行生物安全性和滴度检测，结果发现生物安全性良好。 2.4 转基因细胞系的建立及纯化小鼠胚胎干细胞经Nanog-hrGFP病毒浓缩液连续转导3次后，荧光显微镜观察克隆中部分细胞表达绿色荧光逐渐增多，细胞传代扩增后加入1-5 mg/L的Puro进行筛选，连续筛选12 d，得到了较纯的小鼠胚胎干细胞转基因细胞系。然后再用胰酶消化成单个细胞，待克隆长大后挑取单个克隆进行培养，扩增，即可得到单细胞克隆的小鼠胚胎干细胞转基因细胞系，表达绿色荧光蛋白，荧光显微镜可观察小鼠胚胎干细胞全部为绿色(图6)，表达外源基因导入成功。 2.5 Nanog-hrGFP转基因细胞系的鉴定碱性磷酸酶、Nanog、SSEA-1在增殖活跃的胚胎干细胞中活性较高，常用作原始未分化细胞的标志之一。镜下见小鼠胚胎成纤维细胞不能被染色，Nanog-hrGFP转基因细胞系进行碱性磷酸酶，SSEA-1和Nanog染色为强阳性(图7)。 2.6 转基因细胞系分化过程中Nanog的表达变化Nanog-hrGFP-mES经拟胚体后进行贴壁2，4 d时荧光观察拍照记录，结果发现随着贴壁时间的延长，克隆细胞逐渐散开，转基因细胞逐渐分化成梭形或扁平状，绿色荧光表达降低表明Nanog表达量逐渐降低(图8)。同时qPCR提取检测Nanog的表达情况发现，转基因细胞组与对照组小鼠胚胎干细胞的Nanog表达量也逐渐降低，但实验组表达量降低的更为迅速，也再次证明外源基因Nanog-hrGFP导入成功。"

References

[1] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4): 663-676.

[2] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5): 861-872.

[3] Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858): 1917-1920.

[4] Cheng F, Ke Q, Chen F, et al. Protecting against wayward human induced pluripotent stem cells with a suicide gene. Biomaterials. 2012;33(11): 3195-3204.

[5] 张杨,李秀兰.诱导性多潜能干细胞的安全性及临床应用[J].中国组织工程研究, 2013,17(27):5087-5093.

[6] Li W, Xiang AP. Safeguarding clinical translation of pluripotent stem cells with suicide genes. Organogenesis. 2013;9(1):34-39.

[7] 姚欣鹏,徐旸,张璐,等.分子影像学方法示踪胚胎干细胞移植治疗急性肝损伤[J].中国组织工程研究,2013,17(36): 6481-6488.

[8] Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem. 1989;58: 913-949.

[9] Furuya M, Yasuchika K, Mizutani K, et al. Electroporation of cynomolgus monkey embryonic stem cells. Genesis. 2003;37(4): 180-187.

[10] Ueda S, Yoshikawa M, Ouji Y, et al. Cynomolgus monkey embryonic stem cell lines express green fluorescent protein. J Biosci Bioeng. 2006;102(1): 14-20.

[11] Suter DM, Cartier L, Bettiol E, et al. Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors. Stem Cells. 2006;24(3): 615-623.

[12] Belfield EJ, Hughes RK, Tsesmetzis N, et al. The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence. Nucleic Acids Res. 2007;35(4): 1322-1332.

[13] Chen QJ, Zhou HM, Chen J, et al. A Gateway-based platform for multigene plant transformation. Plant Mol Biol. 2006;62(6): 927-936.

[14] Sivagnanam S, Majumdar A, Yoshimoto K, et al. Early experiences in developing and managing the neuroscience gateway. Concurr Comput. 2015;27(2): 473-488.

[15] Wu TM, Lin KC, Liau WS, et al. A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.). Plant Mol Biol. 2016;90(1-2): 107-115.

[16] Griffin TZ, Kang W, Ma Y, et al. The HAND Database: a gateway to understanding the role of HIV in HIV-associated neurocognitive disorders. BMC Med Genomics. 2015;8(1):70.

[17] Silva J, Nichols J, Theunissen TW, et al. Nanog is the gateway to the pluripotent ground state. Cell, 2009; 138(4): 722-737.

[18] Galvagni F, Neri F.Snail represses Nanog to promote embryonic stem cell differentiation. Genom Data. 2015; 4:82-83.

[19] 沈洁,李金辉,阮静,等.诱导多能干细胞建系及体外造血祖细胞定向分化体系的建立[J].中国组织工程研究,2014, 18(19): 3005-3011.

[20] Evans M. Discovering pluripotency: 30 years of mouse embryonic stem cells. Nat Rev Mol Cell Biol. 2011; 12(10): 680-686.

[21] Kiuru M, Boyer JL, O'Connor TP, et al. Genetic control of wayward pluripotent stem cells and their progeny after transplantation. Cell Stem Cell. 2009;4(4): 289-300.

[22] Wilkinson AC, Goode DK, Cheng YH, et al. Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses. Biol Open. 2013;2(11):1229-1238.

[23] Xia X, Zhang Y, Zieth CR, et al. Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner. Stem Cells Dev. 2007;16(1): 167-176.

[24] 蔡弢艺,陈雄生,贾连顺,等.碱性成纤维细胞生长因子重组腺病毒转染兔骨髓间充质干细胞的表型变化[J].中国组织工程研究,2014,18(23):3727-3731.

[25] Smith-Arica JR, Thomson AJ, Ansell R, et al. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning Stem Cells. 2003;5(1): 51-62.

[26] Prouty AM, Fischer J, Purdom A, et al. Spiritual Coping: A Gateway to Enhancing Family Communication During Cancer Treatment. J Relig Health. 2016;55(1): 269-287.

[27] Amaya CN, Bryan BA. Enrichment of the embryonic stem cell reprogramming factors Oct4, Nanog, Myc, and Sox2 in benign and malignant vascular tumors. BMC Clin Pathol. 2015;15:18.

[28] Kang J, Shakya A, Tantin D. Stem cells, stress, metabolism and cancer: a drama in two Octs. Trends Biochem Sci. 2009;34(10):491-499.

[29] Liedtke S, Stephan M, Kögler G. et al. Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research. Biol Chem. 2008;389(7): 845-850.

[30] Tsai LL, Hu FW, Lee SS, et al. Oct4 mediates tumor initiating properties in oral squamous cell carcinomas through the regulation of epithelial-mesenchymal transition. PLoS One. 2014;9(1):e87207.

[31] Liu J, Wang L, Ma L, et al. Significantly increased expression of OCT4 and ABCG2 in spheroid body-forming cells of the human gastric cancer MKN-45 cell line. Oncol Lett. 2013;6(4):891-896.

[32] Silva J, Nichols J, Theunissen TW, et al. Nanog is the gateway to the pluripotent ground state. Cell. 2009; 138(4): 722-737.

[33] Li WZ, Ai ZY, Wang ZW, et al. GATA-1 directly regulates Nanog in mouse embryonic stem cells. Biochem Biophys Res Commun. 2015;465(3):575-579.

[34] Guo C, Xue Y, Yang G, et al. Nanog RNA binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells. Cell Biol Int. 2015. [Epub ahead of print]