Construction of a high efficient pancreatic duodenal homeobox 1/insulin dual-reporter vector and its preliminary application

doi:10.3969/j.issn.2095-4344.2017.12.017

Abstract

Abstract:

BACKGROUND: Islet beta cell replacement therapy is one of the most promising approaches for treating type 1 diabetes mellitus. However, its large scale application is hampered by a shortage of islet beta cells for transplantation. Pluripotent stem cells are one of ideal seed cells for islet beta cell replacement therapy, but pancreatic beta-cell differentiation is time-consuming and labor-intensive.
OBJECTIVE: To construct a high efficient pancreatic and duodenal homeobox 1 (Pdx1)/insulin dual-reporter vector and to monitor the key genes expression during pancreatic beta-cell differentiation from pluripotent stem cells.
METHODS: In order to construct a high efficient Pdx1/insulin dual-reporter vector, puromycin resistance gene was firstly introduced into pTiger vector, and then the original 410 bp mouse Ins1 promoter of the vector was replaced by 646 bp mouse Ins1 promoter. Finally, the dual-reporter vector was transduced into INS-1 and human induced pluripotent stem cells to testify its function.
RESULTS AND CONCLUSION: The high efficient Pdx1/insulin dual-reporter vector was constructed successfully. The vector successfully acquired puromycin resistance gene and high gene expression efficacy of insulin in INS-1 cells. The specific gene expression pattern of Pdx1/insulin was first found in INS-1 cells. To conclude, the real-time monitoring function of Pdx1/insulin expression is preliminarily confirmed during pancreatic beta-cell differentiation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Multipotent Stem Cells, Insulin, Genes, Reporter, Tissue Engineering

CLC Number:

R318

Ye Ling-ling1, Li Shi-chong1, Sun Hai-yan2, Lan San-chun3, Chen Zhao-lie1, Wang Qi-wei1. Construction of a high efficient pancreatic duodenal homeobox 1/insulin dual-reporter vector and its preliminary application[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(12): 1903-1908.

Figures/Tables 1

2.1 Pdx1/Insulin双报告基因载体的构建首先，在pTiger-Pdx1::mRFP-Ins1::EGFP载体中引入嘌呤霉素抗性，通过PCR成功地从pLKO.1载体上扩增了1 236 bp的hPGK-Puromycin片断(图1A)。hPGK-Puromycin片断和pTiger载体分别经PmeⅠ/NotⅠ双酶切，胶回收纯化后，用T4 DNA连接酶将目的片断连入pTiger载体。经质粒转化、涂板后，随机挑取6个克隆进行PCR鉴定，共获得4个阳性克隆(图1B)。4个阳性克隆经PmeⅠ/NotⅠ双酶切进一步鉴定后，获得1个阳性克隆(图1C)。经测序证明此克隆序列完全正确。为提高pTiger载体的insulin表达效率，首先从p-Ins1::hrGFP载体上通过PCR成功地扩增了1 411 bp的Ins1-hrGFP片断(图2A)，Ins1-hrGFP片断和pTiger载体分别经NheⅠ/PemⅠ双酶切，胶回收纯化后，用T4 DNA连接酶将目的片断连入pTiger载体。经质粒转化、涂板后，随机挑取4个克隆进行PCR鉴定，共获得3个阳性克隆(图2B)。3个阳性克隆经NheⅠ/PemⅠ双酶切进一步鉴定后，获得2个阳性克隆(图2C)。经测序证明2个克隆的序列完全正确。至此，成功地在原来pTiger载体上引入嘌呤霉素抗性，并以Ins1-hrGFP替换原来的Ins1-EGFP，构建高效的pTiger-Pdx1::mRFP-Ins1::hrGFP- hPGK::Puromycin载体(图3)。 2.2 双报告基因载体在INS-1中的表达通过观察细胞内荧光蛋白的表达，验证双报告基因载体在细胞内的功能。INS-1细胞固有表达Pdx1和insulin基因。病毒感染后24 h，细胞内开始有红色荧光蛋白(RFP)和绿色荧光蛋白(GFP)表达，提示细胞表达Pdx1和insulin基因。48-72 h细胞内荧光蛋白表达强度逐渐增强，而后荧光强度无显著变化。病毒感染后4 d，约有15%的细胞无红色荧光蛋白和绿色荧光蛋白表达，这可能是由于病毒感染效率所致。随后对细胞进行加压筛选，在细胞培养基中添加1 mg/L嘌呤霉素，正常未感染病毒的INS-1细胞作为筛选对照组。三四天后对照组细胞全部死亡，而病毒感染细胞由于表达嘌呤霉素抗性基因，细胞能够正常生长，只是细胞增殖速度有所减缓(图4)。依据细胞内荧光蛋白信号强度表达的差异可将INS-1细胞分为3种类型：Pdx1/insulin高表达(Pdx1highinsulinhigh)、Pdx1/Insulin中等表达(Pdx1mediuminsulinmedium)和Pdx1低表达/insulin低或无表达(Pdx1lowinsulin-)(图4)。"

References

[1] Xu Y, Wang L, He J, et al. Prevalence and control of diabetes in Chinese adults. JAMA. 2013;310(9):948-959.

[2] Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet. 2001;358(9277):221-229.

[3] Sachs DH, Bonner-Weir S. New islets from old. Nat Med. 2000;6(3):250-251.

[4] Serup P, Madsen OD, Mandrup-Poulsen T. Islet and stem cell transplantation for treating diabetes. BMJ. 2001;322(7277):29-32.

[5] Xie M, Ye H, Wang H, et al. β-cell-mimetic designer cells provide closed-loop glycemic control. Science. 2016;354 (6317):1296-1301.

[6] Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science. 2001;292(5520):1389-1394.

[7] Soria B, Roche E, Berná G, et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes. 2000;49(2): 157-162.

[8] Assady S, Maor G, Amit M, et al. Insulin production by human embryonic stem cells. Diabetes. 2001;50(8):1691-1697.

[9] Moritoh Y, Yamato E, Yasui Y, et al. Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes. 2003;52(5):1163-1168.

[10] Lester LB, Kuo HC, Andrews L, et al. Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes. Reprod Biol Endocrinol. 2004;2:42.

[11] León-Quinto T, Jones J, Skoudy A, et al. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia. 2004;47(8):1442-1451.

[12] D'Amour KA, Bang AG, Eliazer S, et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 2006;24(11): 1392-1401.

[13] Kroon E, Martinson LA, Kadoya K, et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol. 2008;26(4):443-452.

[14] Maehr R, Chen S, Snitow M, et al. Generation of pluripotent stem cells from patients with type 1 diabetes. Proc Natl Acad Sci U S A. 2009;106(37):15768-15773.

[15] Pagliuca FW, Millman JR, Gürtler M, et al. Generation of functional human pancreatic β cells in vitro. Cell. 2014; 159(2): 428-439.

[16] 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.

[17] 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.

[18] Kim JB, Greber B, Araúzo-Bravo MJ, et al. Direct reprogramming of human neural stem cells by OCT4. Nature. 2009;461(7264):649-653.

[19] Moriguchi H, Chung RT, Mihara M, et al. Generation of human induced pluripotent stem cells from liver progenitor cells by only small molecules. Hepatology. 2010;52(3):1169.

[20] Giorgetti A, Montserrat N, Rodriguez-Piza I, et al. Generation of induced pluripotent stem cells from human cord blood cells with only two factors: Oct4 and Sox2. Nat Protoc. 2010;5(4): 811-820.

[21] Shaer A, Azarpira N, Karimi MH. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection. Appl Biochem Biotechnol. 2014;174(1):242-258.

[22] Prasain N, Lee MR, Vemula S, et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat Biotechnol. 2014;32(11):1151-1157.

[23] Diekmann U, Lenzen S, Naujok O. A reliable and efficient protocol for human pluripotent stem cell differentiation into the definitive endoderm based on dispersed single cells. Stem Cells Dev. 2015;24(2):190-204.

[24] Toyoda T, Mae S, Tanaka H, et al. Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells. Stem Cell Res. 2015;14(2):185-197.

[25] Ikonomou L, Kotton DN. Derivation of Endodermal Progenitors From Pluripotent Stem Cells. J Cell Physiol. 2015; 230(2):246-258.

[26] Shaer A, Azarpira N, Vahdati A, et al. Differentiation of human-induced pluripotent stem cells into insulin-producing clusters. Exp Clin Transplant. 2015;13(1):68-75.

[27] Mauda-Havakuk M, Litichever N, Chernichovski E, et al. Ectopic PDX-1 expression directly reprograms human keratinocytes along pancreatic insulin-producing cells fate. PLoS One. 2011;6(10):e26298.

[28] Zhou Q, Brown J, Kanarek A, et al. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature. 2008; 455(7213):627-632.

[29] Akinci E, Banga A, Greder LV, et al. Reprogramming of pancreatic exocrine cells towards a beta (β) cell character using Pdx1, Ngn3 and MafA. Biochem J. 2012;442(3): 539-550.

[30] Shi Y, Hou L, Tang F, et al. Inducing embryonic stem cells to differentiate into pancreatic beta cells by a novel three-step approach with activin A and all-trans retinoic acid. Stem Cells. 2005;23(5):656-662.

[31] Kunisada Y, Tsubooka-Yamazoe N, Shoji M, et al. Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells. Stem Cell Res. 2012;8(2):274-284.

[32] Loh KM, Ang LT, Zhang J, et al. Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell Stem Cell. 2014; 14(2):237-252.

[33] Rezania A, Bruin JE, Arora P, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. 2014;32(11):1121-1133.

[34] Wang Q, Wang H, Sun Y, et al. The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation. J Cell Sci. 2013;126(Pt 16):3638-3648.

[35] Murtaugh LC, Melton DA. Genes, signals, and lineages in pancreas development. Annu Rev Cell Dev Biol. 2003;19: 71-89.

[36] Jensen J. Gene regulatory factors in pancreatic development. Dev Dyn. 2004;229(1):176-200.

[37] Szabat M, Luciani DS, Piret JM, et al. Maturation of adult beta-cells revealed using a Pdx1/insulin dual-reporter lentivirus. Endocrinology. 2009;150(4):1627-1635.