|
|
|
| Construction of the IRES-based Vector for Multiple Gene Co-expression |
| TIAN Cong-hui1, XIE Xue-mei1, LI Ying1, YIN Xiao-dong2, HAN Jun1, LI Jun1 |
1. School of Pharmacy and Institute of BioPharmaceutical Rescarch, Liaocheng University, Liaocheng 252000, China;
2. Yixing Cell Biotechnology Limited Company, Yixing 214200, China |
|
|
|
Abstract Objective: An IRES-based vector was constructed to achieve co-expression of two target genes with the screening marker gene promoted by the single promoter, and to improve the screening efficiency of multiple genes co-stable expression cell lines. Methods: A bicistronic expression element BamHI-MCS1-IRES-MCS2-IRES-BsiWI which has two multiple cloning sites was designed and synthesized. The vector named pLV-2MCS-Puro was constructed by inserting the element into the skeleton vector pLV-MCS-Puro which was constructed previously in lab. The DsRed2 and EGFP genes were inserted simultaneously into the vector to test the screening efficiency of multiple genes co-stable expression cell lines. Results: The vector pLV-2MCS-Puro and the recombinant plasmid pLV-DsRed2-EGFP-Puro were constructed successfully. Transient transfection experiment showed that the vector can mediate co-expression of multiple genes. MDCK and HeLa cell pools resistant to puromycin were obtained through transfection of the recombinant plasmid. The fluorescent inverted microscope showed that DsRed2 gene at the upstream of the IRES sequence and EGFP gene at the downstream of IRES sequence were co-expressed in cells, and the double positive rate was close to 100%. It indicated that this vector has high screening efficiency. The results of genomic PCR, RT-PCR and Western blot showed that DsRed2 and EGFP genes were stably integrated into cell genome and the two proteins were expressed consistently. Conclusion: The IRES-based vector pLV-2MCS-Puro was successfully constructed and proved to be efficiently in screening multiple genes co-stable expression cell lines. This vector will have certain application prospects in studying protein interactions and constructing engineering cell lines.
|
|
Received: 07 February 2017
Published: 25 July 2017
|
|
|
|
|
[1] Allera-Moreau C, Chomarat P, Audinot V, et al. The use of IRES-based bicistronic vectors allows the stable expression of recombinant G-protein coupled receptors such as NPY5 and histamine 4. Biochimie, 2006, 88(6):737-746.
[2] Pelletier J,Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature, 1988, 334(6180):320-325.
[3] Jang S K, Krausslich H G, Nicklin M J, et al. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. Journal of Virology, 1988, 62(8):2636-2643.
[4] Lewis S M, Holcik M. For IRES trans-acting factors, it is all about location. Oncogene, 2008, 27(8):1033-1035.
[5] Lu J, Zhang J, Lin M, et al. IRES: translation element of RNA viruses. Chinese Journal of Biochemistry & Molecular Biology, 2007, 27(03):513-518.
[6] Jackson R J, Hellen C U, Pestova T V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Reviews. Molecular Cell Biology, 2010, 11(2):113-127.
[7] Chappell S A, Edelman G M, Mauro V P. Ribosomal tethering and clustering as mechanisms for translation initiation. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(48):18077-18082.
[8] Shatsky I N, Dmitriev S E, Terenin I M, et al. Cap- and IRES-independent scanning mechanism of translation initiation as an alternative to the concept of cellular IRESs. Molecules and Cells, 2010, 30(4):285-293.
[9] Komar A A, Hatzoglou M. Internal ribosome entry sites in cellular mRNAs: mystery of their existence. Journal of Biological Chemistry, 2005, 280(25):23425-23428.
[10] Misteli T, Spector D L. Applications of the green fluorescent protein in cell biology and biotechnology. Nature Biotechnology, 1997, 15(10):961-964.
[11] Fussenegger M, Moser S, Bailey J E. pQuattro vectors allow one-step multigene metabolic engineering and auto-selection of quattrocistronic artificial mammalian operons. Cytotechnology, 1998, 28(1-3):229-235.
[12] Martinez-Salas E, Lopez de Quinto S, Ramos R, et al. IRES elements: features of the RNA structure contributing to their activity. Biochimie, 2002, 84(8):755-763.
[13] Martinez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Current Opinion in Biotechnology, 1999, 10(5):458-464.
[14] Liu X, Constantinescu S N, Sun Y, et al. Generation of mammalian cells stably expressing multiple genes at predetermined levels. Analytical Biochemistry, 2000, 280(1):20-28.
[15] Murakami M, Watanabe H, Niikura Y, et al. High-level expression of exogenous genes by replication-competent retrovirus vectors with an internal ribosomal entry site. Gene, 1997, 202(1-2):23-29.
[16] Rayssac A, Neveu C, Pucelle M, et al. IRES-based vector coexpressing FGF2 and Cyr61 provides synergistic and safe therapeutics of lower limb ischemia. Molecular Therapy, 2009, 17(12):2010-2019.
[17] Jazwa A, Tomczyk M, Taha H M, et al. Arteriogenic therapy based on simultaneous delivery of VEGF-A and FGF4 genes improves the recovery from acute limb ischemia. Vasc Cell, 2013, 5(1):13.
[18] Zhang C, Wang K Z, Qiang H, et al. Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo. Acta Pharmacologica Sinica, 2010, 31(7):821-830.
[19] Ngoi S M, Chien A C, Lee C G. Exploiting internal ribosome entry sites in gene therapy vector design. Current Gene Therapy, 2004, 4(1):15-31.
[20] Cepko C L, Roberts B E, Mulligan R C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell, 1984, 37(3):1053-1062.
[21] Korman A J, Frantz J D, Strominger J L, et al. Expression of human class Ⅱ major histocompatibility complex antigens using retrovirus vectors. Proceedings of the National Academy of Sciences of the United States of America, 1987, 84(8):2150-2154.
[22] Fallot S, Ben Naya R, Hieblot C, et al. Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production. Nucleic Acids Research, 2009, 37(20):e134.
[23] Doronina V A, Wu C, de Felipe P, et al. Site-specific release of nascent chains from ribosomes at a sense codon. Molecular and Cellular Biology, 2008, 28(13):4227-4239.
[24] de Felipe P, Luke G A, Hughes L E, et al. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends in Biotechnology, 2006, 24(2):68-75.
[25] Bosch M K, Nerbonne J M, Ornitz D M. Dual transgene expression in murine cerebellar Purkinje neurons by viral transduction in vivo. PLoS ONE, 2014, 9(8):e104062.
[26] Saleh L, Perler F B. Protein splicing in cis and in trans. The Chemical Record, 2006, 6(4):183-193.
[27] Eryilmaz E, Shah N H, Muir T W, et al. Structural and dynamical features of inteins and implications on protein splicing. Journal of Biological Chemistry, 2014, 289(21):14506-14511.
[28] Renaud-Gabardos E, Hantelys F, Morfoisse F, et al. Internal ribosome entry site-based vectors for combined gene therapy. World Journal of Experimental Medicine, 2015, 5(1):11-20.
[29] Alleramoreau C, Dellucclavières A, Castano C, et al. Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle. BMC Biotechnology, 2007, 7(1):1-12.
[30] Chu C, Lugovtsev V, Golding H, et al. Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(35):14802-14807. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
