Construction of Lentiviral Vector Containing Murine Vascular Endothelial Growth Factor Gene and Its Expression in NS-1 Murine Myeloma Cell Line_West China Medical Journal

Authors：

YANG Jinrong ^1,2 , ZENG Ke ^1,2 , LI Jun ¹ ,  NIU Ting ^1,2

1. Department of Hematology and Research Laboratory of Hematology, West China Hospital, Sichuan University; 2. State Key Laboratory of Biotherapy, Sichuan University; Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

NIU Ting, Email: 499392105@qq.com

Keywords：

小鼠血管内皮生长因子; 慢病毒载体; NS-1细胞株; 多发性骨髓瘤

DOI：

10.7507/1002-0179.20130310

Video：

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目的　构建含小鼠血管内皮生长因子（mVEGF）的重组慢病毒表达载体，包装成病毒颗粒后感染NS-1小鼠骨髓瘤细胞株，以便进一步探索VEGF在骨髓瘤病理生理机制中的作用。方法　聚合酶链反应法扩增mVEGF基因，克隆入含嘌呤霉素抗性的pCDH慢病毒表达载体，构建出表达mVEGF的慢病毒表达载体pCDH-mVEGF；采用磷酸钙法将慢病毒系统三质粒pCDH-mVEGF、psPAX2、pMD2.G共转染293FT细胞包装病毒，分别收集转染后48 h和72 h病毒上清并感染靶细胞NS-1，初次感染72 h后开始采用嘌呤霉素筛选稳定株，筛选2周后采用ELISA法检测稳定株细胞培养上清中mVEGF的表达，建立出稳定高表达mVEGF的NS-1小鼠骨髓瘤细胞株。结果　成功构建重组慢病毒表达质粒pCDH-mVEGF，并包装成慢病毒颗粒，感染NS-1细胞株后获得靶基因的稳定高表达。结论　成功构建出含mVEGF的慢病毒表达载体pCDH-mVEGF，慢病毒系统能有效介导目的基因在NS-1小鼠骨髓瘤细胞株中稳定表达，病毒包装成功并能有效感染NS-1细胞，为进一步探索VEGF在骨髓瘤病理生理机制中的作用奠定了基础。

Citation： YANG Jinrong,ZENG Ke,LI Jun,NIU Ting,.. Construction of Lentiviral Vector Containing Murine Vascular Endothelial Growth Factor Gene and Its Expression in NS-1 Murine Myeloma Cell Line. West China Medical Journal, 2013, 28(7): 998-1002. doi: 10.7507/1002-0179.20130310 Copy

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2.	Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development[J]. Nature, 2005, 438 (7070): 937-945.
3.	Houck KA, Ferrara N, Winer J, et al. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA[J]. Mol Endocrinol, 1991, 5(12): 1806-1814.
4.	Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature, 1996, 380(6573): 439-442.
5.	Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J]. Nature, 1996, 380(6573): 435-439.
6.	Senger DR, Connolly DT, Van de Water L, et al. Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor[J]. Cancer Res, 1990, 50(6): 1774-1778.
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11.	Aguayo A, Kantarjian H, Manshouri T, et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes[J]. Blood, 2000, 96(6): 2240-2245.
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14.	Gerber HP, Malik AK, Solar GP, et al. VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism[J]. Nature, 2002, 417(6892): 954-958.
15.	Gabrilovich DI, Chen HL, Girgis KR, et al. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells[J]. Nat Med, 1996, 2(10): 1096-1103.
16.	Nakagawa M, Kaneda T, Arakawa T, et al. Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts[J]. FEBS Lett, 2000, 473(2): 161-164.
17.	Henriksen K, Karsdal M, Delaisse JM, et al. RANKL and vascular endothelial growth factor (VEGF) induce osteoclast chemotaxis through an ERK1/2-dependent mechanism[J]. Journal of Biological Chemistry, 2003, 278(49): 48745-48753.
18.	Tiscornia G, Singer O, Verma IM. Production and purification of lentiviral vectors[J]. Nat Protoc, 2006, 1(1): 241-245.
19.	Podar K, Anderson KC. The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications[J]. Blood, 2005, 105(4): 1383-1395.
20.	Paesler J, Gehrke I, Poll-Wolbeck SJ, et al. Targeting the vascular endothelial growth factor in hematologic malignancies[J]. Eur J Haematol, 2012, 89(5): 373-384.
21.	Hattori K, Dias S, Heissig B, et al. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells[J]. J Exp Med, 2001, 193(9): 1005-1014.
22.	Melder RJ, Koenig GC, Witwer BP, et al. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium[J]. Nat Med, 1996, 2(9): 992-997.
23.	Barleon B, Sozzani S, Zhou D, et al. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1[J]. Blood, 1996, 87(8): 3336-3343.
24.	Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth[J]. Nat Med, 2001, 7(11): 1194-1201.
25.	Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J]. Science, 1996, 272(5259): 263-267.
26.	Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics[J]. Nat Med, 2001, 7(1): 33-40.
27.	Breckpot K, Aerts JL, Thielemans K. Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics[J]. Gene Ther, 2007, 14(11): 847-862.

1. 　Neufeld G, Cohen T, Gengrinovitch S, et al. Vascular endothelial growth factor (VEGF) and its receptors [J]. FASEB J, 1999, 13 (1): 9-22.
2. 　Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development[J]. Nature, 2005, 438 (7070): 937-945.
3. 　Houck KA, Ferrara N, Winer J, et al. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA[J]. Mol Endocrinol, 1991, 5(12): 1806-1814.
4. 　Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature, 1996, 380(6573): 439-442.
5. 　Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J]. Nature, 1996, 380(6573): 435-439.
6. 　Senger DR, Connolly DT, Van de Water L, et al. Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor[J]. Cancer Res, 1990, 50(6): 1774-1778.
7. 　Folkman J. Tumor angiogenesis: therapeutic implications[J]. N Engl J Med, 1971, 285(21): 1182-1186.
8. 　Vacca A, Ribatti D, Roncali L, et al. Angiogenesis in B cell lymphoproliferative diseases[J]. Leuk Lymphoma, 1995, 20(1-2): 27-38.
9. 　Bellamy WT. Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies[J]. Semin Oncol, 2001, 28(6): 551-559.
10. 　Bellamy WT, Richter L, Frutiger Y, et al. Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies[J]. Cancer Res, 1999, 59(3): 728-733.
11. 　Aguayo A, Kantarjian H, Manshouri T, et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes[J]. Blood, 2000, 96(6): 2240-2245.
12. 　Hussong JW, Rodgers GM, Shami PJ. Evidence of increased angiogenesis in patients with acute myeloid leukemia[J]. Blood, 2000, 95(1): 309-313.
13. 　Lundberg LG, Lerner R, Sundelin P, et al. Bone marrow in polycythemia vera, chronic myelocytic leukemia, and myelofibrosis has an increased vascularity[J]. Am J Pathol, 2000, 157(1): 15-19.
14. 　Gerber HP, Malik AK, Solar GP, et al. VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism[J]. Nature, 2002, 417(6892): 954-958.
15. 　Gabrilovich DI, Chen HL, Girgis KR, et al. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells[J]. Nat Med, 1996, 2(10): 1096-1103.
16. 　Nakagawa M, Kaneda T, Arakawa T, et al. Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts[J]. FEBS Lett, 2000, 473(2): 161-164.
17. 　Henriksen K, Karsdal M, Delaisse JM, et al. RANKL and vascular endothelial growth factor (VEGF) induce osteoclast chemotaxis through an ERK1/2-dependent mechanism[J]. Journal of Biological Chemistry, 2003, 278(49): 48745-48753.
18. 　Tiscornia G, Singer O, Verma IM. Production and purification of lentiviral vectors[J]. Nat Protoc, 2006, 1(1): 241-245.
19. 　Podar K, Anderson KC. The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications[J]. Blood, 2005, 105(4): 1383-1395.
20. 　Paesler J, Gehrke I, Poll-Wolbeck SJ, et al. Targeting the vascular endothelial growth factor in hematologic malignancies[J]. Eur J Haematol, 2012, 89(5): 373-384.
21. 　Hattori K, Dias S, Heissig B, et al. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells[J]. J Exp Med, 2001, 193(9): 1005-1014.
22. 　Melder RJ, Koenig GC, Witwer BP, et al. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium[J]. Nat Med, 1996, 2(9): 992-997.
23. 　Barleon B, Sozzani S, Zhou D, et al. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1[J]. Blood, 1996, 87(8): 3336-3343.
24. 　Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth[J]. Nat Med, 2001, 7(11): 1194-1201.
25. 　Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J]. Science, 1996, 272(5259): 263-267.
26. 　Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics[J]. Nat Med, 2001, 7(1): 33-40.
27. 　Breckpot K, Aerts JL, Thielemans K. Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics[J]. Gene Ther, 2007, 14(11): 847-862.

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