Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmidsReport as inadecuate




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BMC Research Notes

, 7:626

First Online: 10 September 2014Received: 23 January 2014Accepted: 05 September 2014DOI: 10.1186-1756-0500-7-626

Cite this article as: Liu, X., Ping, H. & Zhang, C. BMC Res Notes 2014 7: 626. doi:10.1186-1756-0500-7-626

Abstract

BackgroundStable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 AAV2 Rep protein is known to induce rAAV DNA integration into a specific site AAVS1 of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line.

FindingsWe have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII FVIII-BDD and GFP gene flanked by AAV ITRs. Human embryonic kidney HEK 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity.

ConclusionWe developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.

KeywordsB-domain-deleted FVIII FVIII-BDD Site-specific integration HEK 293 cell line Adeno-associated virus AAV AbbreviationsrFVIIIRecombinant factor VIII

rhFVIIIRecombinant human factor VIII

FVIII-BDDB-domain-deleted FVIII

AAV2Adeno-associated virus type 2

ITRInverted terminal repeat

HEK 293Human embryonic kidney293.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-7-626 contains supplementary material, which is available to authorized users.

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Author: Xiaomei Liu - Han Ping - Chun Zhang

Source: https://link.springer.com/



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