建立以重組蛋白生產安全之自體多能性幹細胞的方法

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趙冠豪(Guan-Hao Zhao) 查詢紙本館藏

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論文名稱

建立以重組蛋白生產安全之自體多能性幹細胞的方法
(Formulating a protocol for generating safe pluripotent stem cells from somatic cells by recombinant proteins)

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摘要(中)

利用特定轉錄因子將體細胞轉變為誘導多能性幹細胞在再生醫學上有令人期待的前景。至今已有許多方法能產生誘導多能性幹細胞，例如使用反轉錄病毒、慢病毒、質體、轉位子、合成信使核醣核酸以及重組蛋白。然而，外送的基因物質可能造成不預期的基因組突變。相較之下，以送入重組蛋白產生多能性幹細胞的方式可以避免去氧核醣核酸隨機插入的可能性。在這篇研究中，我們利用聚精胺酸蛋白轉導區塊來送達重組的重新編成因子。五個重新編成因子：Oct4,Sox2,Nanog,Lin28和c-Myc的羧基端都黏合上聚精胺酸蛋白轉導區塊。我們能將這些因子順利的表達並純化；然而，Oct4和Nanog蛋白的表現量相對來說非常低。令我們驚訝的是，將MyoD轉活區塊黏合在Oct4和Nanog的氨基端顯著性的增加蛋白表現。這些蛋白表現在大腸桿菌的包涵體或是原生型態都可以被溶解、重新摺疊並以鎳螯合物凝膠純化。純化的蛋白能轉導通過細胞膜並轉移至細胞核。現今我們正以這些蛋白組合一些小分子化合物，如丙戊酸，來測定最適合重新編成體細胞轉變為誘導多能性幹細胞以及種系特異性幹細胞的條件。

摘要(英)

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined factors has showed hopeful perspective in regenerative medicine. Currently, many approaches are employed to generate iPSCs, including retroviruses, lentiviruses, adenoviruses, plasmids, transposons, synthetic mRNAs and recombinant proteins; however, the introduction of ectopic genetic materials may cause unexpected genetic modifications. In contrast, delivery of recombinant proteins can avoid the possibility of random DNA integration. In this study, we utilized poly-arginine (poly-R) protein transduction domain (PTD) for delivery of recombinant reprogramming factors. The PTD was fused to the C-terminus of five reprogramming factors: Oct4, Sox2, Nanog, Lin28 and c-Myc. These chimera factors could be expressed and purified with normal function in E.coli; however, the expression of Oct4 and Nanog was relatively low. To our surprise, fusion of the powerful MyoD transactivation domain to the N-terminus of Oct4 and Nanog significantly enhanced the protein yield. These proteins expressed in E.coli as inclusion bodies or native forms were solubilized, refolded, and purified using Nickel-Chelating Sepharose. These purified proteins were able to transduce the plasma membrane and translocate into nucleus. Currently, we are combining these proteins and small compounds, such as valproic acid, to determine the optimal condition for reprogram somatic cells into iPSC and lineage-specific stem cells.

關鍵字(中)

★ 誘導性多功能幹細胞
★ 蛋白轉導區塊
★ 重新編成

關鍵字(英)

★ induced pluripotent stem cells
★ protein transduction domain
★ Reprogramming

論文目次

Table of Contents
中文摘要 i
Abstract ii
Declaration iii
Acknowledgement iv
Abbreviations v
Table of Contents vi
I. Introduction 1
II. Materials and Methods 8
1. Cloning 8
1-1. Plasmid 8
1-1.1. pET-LpRH, pET-M3-LpRH and pET-HA2-LpRH 8
1-1.2. pET-M3-Oct4-LpRH 8
1-1.3. pET-M3-Nanog-LpRH, pET-HA2-Nanog-LpRH 8
1-1.4. pET-Klf4-LpRH and pET-M3-Klf4-LpRH 8
1-1.5. pET-HA2-p53R248Q-LpRH and pET-HA2-p53R248W-LpRH 9
1-1.6. pGEX4T1-LIF 9
1-1.7. pStable-Fgf4-promoter-enhancer 9
1-2. Molecular cloning technique 9
1-2.1. Polymerase chain reaction 9
1-2.2. Overlap extension polymerase chain reaction (OE-PCR) 10
1-2.3. Enzymatic digestion 10
1-2.4. Blunt-end formation 11
1-2.5. Phosphorylation of insert DNA 11
1-2.6. De-phosphorylation of vector DNA 11
1-2.7. Ligation 11
1-2.8. Transformation 11
1-2.9. Alkaline lysis method for plasmid DNA isolation 12
2. Protein expression and purification 12
2-1. Induction of recombinant protein 12
2-2. Column preparation 13
2-3. Purification of recombinant protein under native condition 14
2-4. Isolation and solubilization of inclusion bodies 14
2-5. Purification of recombinant protein under denature condition 15
2-6. Refolding and Storage 15
3. Cell culture and transfection 16
3-1. Cell line 16
3-2. Stable cell line 16
3-3. Primary culture 17
4. Generation of induced pluripotent stem cells 17
4-1. Feeder cell preparation 17
4-2. iPSCs generation 18
5. Analytical technique 18
5-1. Luciferase assay 18
5-2. Reverse transcription polymerase chain reaction (RT-PCR) analysis 18
5-3. Western blot analysis 19
5-4. Immunofluorescence staining 21
5-5. Trypan blue staining 22
III. Results 23
1. Construction of recombinant reprogramming factor-expressing plasmids 23
2. Expression and purification of recombinant reprogramming factors 25
3. Characterization of recombinant reprogramming factors 29
4. Generation of iPS cells from mouse somatic cells 31
IV. Discussion 33
V. Reference 38
VI. Figures 45
Fig. 1. Cloning strategy of pET-Reprogramming factor-LpRH expressing plasmids. 46
Fig. 2. Cloning strategy of pET-M3-Reprogramming factor-LpRH expressing plasmids. 47
Fig. 3. Accuracy of pET-HA2-LpRH, pET-HA2-p53R248Q and p53R248W-LpRH and pGEX4T1-LIF 49
Fig. 4. Protein expression of RFs in Rosetta. 50
Fig. 5. Solubility of RFs overexpressed in Rosetta. 51
Fig. 6. Comparison of strategy 1 and strategy 2 in purifying M3O. 52
Fig. 7. Purification of six RFs. 54
Fig. 8. Avoiding aggregate formation during dialysis. 55
Fig. 9. Purification of p53R248Q, p53R248W and GST-LIF. 56
Fig. 10. Characterization of protein translocation of M3O in C3H10T1/2 by immunofluorescence. 57
Fig. 11. Characterization of 12R-mediated translocation of LpRH-containing proteins in C3H10T1/2. 59
Fig. 12. Live cell analysis of LpRH-mediated protein transduction. 60
Fig. 13. Reprogramming somatic cells using RFs. 62
VII. Appendixes 63
Appendix 1：Supplementary figures 63
Fig. S1. Stem cell hierarchy. 63
Fig. S2. Factor delivery methods for iPSC derivation. 64
Fig. S3. Pathway for the transduction of R9 into cells. 64
Fig. S4. Using luciferase assay to monitor the transcriptional activity of RFs. 66
Fig. S5. Using electrophoresis mobility shift assay to analyze DNA binding activity of M3O and Sox2 on Fgf4 enhancer. 67
Appendix 2：Plasmid and primer list 68
Appendix 3：Length and molecular weight of the recombinant proteins 73
Appendix 4：Solutions 74

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2014-7-29

推文