Generation of iPS Cells Using a BacMam Multigene Expression System

Takata, Yoko; Kishine, Hiroe; Sone, Takefumi; Andoh, Taichi; Nozaki, Masami; Poderycki, Michael J.; Chesnut, Jonathan D.; Imamoto, Fumio

doi:10.1247/csf.11008

Cited by 20 publications

(16 citation statements)

References 53 publications

(73 reference statements)

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“…The data presented here is in agreement with previously published studies that successfully used F2A sequences of various lengths in in vitro and in vivo heterologous systems [24, 25, 27–29, 31]. To avoid using longer F2A sequences that remain attached at the C-terminus of the upstream protein, researchers have used shorter F2As but have reported a range of cleavage activities for F2As of the same length [9, 12, 24, 27, 28, 39].…”

Section: Discussionsupporting

confidence: 92%

Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues

Minskaia

Ryan

2013

BioMed Research International

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Many biomedical applications absolutely require, or are substantially enhanced by, coexpression of multiple proteins from a single vector. Foot-and-mouth disease virus 2A (F2A) and “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A) are used widely for this purpose since multiple proteins can be coexpressed by linking open reading frames (ORFs) to form a single cistron. The activity of F2A “cleavage” may, however, be compromised by both the use of shorter versions of F2A and the sequences (derived from multiple-purpose cloning sites) used to link F2A to the upstream protein. To characterise these effects, different lengths of F2A and T2A were inserted between green and cherry fluorescent proteins. Mutations were introduced in the linker region immediately upstream of both F2A- and T2A-based constructs and activities determined using both cell-free translation systems and transfected cells. In shorter versions of F2A, activity may be affected by both the C-terminal sequence of the protein upstream and, equally strikingly, the residues immediately upstream introduced during cloning. Mutations significantly improved activity for shorter versions of F2A but could decrease activity in the case of T2A. These data will aid the design of cloning strategies for the co-expression of multiple proteins in biomedical/biotechnological applications.

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Section: Discussionsupporting

confidence: 92%

Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues

Minskaia

Ryan

2013

BioMed Research International

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“…To date, various reporter proteins together with proteins requiring discrete co- and post-translational subcellular localization have been successfully co-expressed in in vitro and in vivo heterologous systems using F2A sequences of various lengths [11,17-24]. However, some limitations of using this strategy were reported.…”

Section: Introductionmentioning

confidence: 99%

Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications

2013

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BackgroundMany biomedical applications require the expression or production of therapeutic hetero-multimeric proteins/protein complexes: in most cases only accomplished by co-ordinated co-expression within the same cell. Foot-and-mouth disease virus 2A (F2A) and ‘2A-like’ sequences are now widely used for this purpose. Since 2A mediates a co-translational ‘cleavage’ at its own C-terminus, sequences encoding multiple proteins (linked via 2As) can be concatenated into a single ORF: a single transgene. It has been shown that in some cases, however, the cleavage efficiency of shorter versions of F2A may be inhibited by the C-terminus of certain gene sequences immediately upstream of F2A. This paper describes further work to optimise F2A for co-expression strategies.ResultsWe have inserted F2A of various lengths in between GFP and CherryFP ‘reporter’ proteins (in reciprocal or tandem arrangements). The co-expression of these proteins and cleavage efficiencies of F2As of various lengths were studied by in vitro coupled transcription and translation in rabbit reticulocyte lysates, western blotting of HeLa cell lysates and fluorescence microscopy.ConclusionsOptimal and suboptimal lengths of F2A sequences were identified as a result of detailed ‘fine-tuning’ of the F2A sequence. Based on our data and the model according to which 2A activity is a product of its interaction with the exit tunnel of the ribosome, we suggest the length of the F2A sequence which is not ‘sensitive’ to the C-terminus of the upstream protein that can be successfully used for co-expression of two proteins for biomedical applications.

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“…For instance, minicircle vector has been used to generate iPS cells (57), but the reprogramming efficiency is limited by the low transfection efficiency for the primary cells. As BV can deliver essential transcription factor genes for the generation of iPS cells (58), it is tempting to use the hybrid BV for highly efficient in situ generation of minicircles containing essential transcription factor genes in primary cells to enhance the reprogramming efficiency. Furthermore, AAV and lentivirus vectors are commonly produced by transfection of producer cells, but the procedures are inefficient and costly.…”

Section: Discussionmentioning

confidence: 99%

Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

Sung¹,

Chen²,

Lin³

et al. 2013

Nucleic Acids Research

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Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40–75%), but approached ≈90–95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein–Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf genes, the efficient recombination/minicircle formation prolonged and enhanced the growth factor expression in hASCs. The prolonged bone morphogenetic protein 2 expression ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this BV vector exploiting Cre-mediated recombination and oriP/EBNA1 conferred remarkably high recombination efficiency, which prolonged and enhanced the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.

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Generation of iPS Cells Using a BacMam Multigene Expression System

Cited by 20 publications

References 53 publications

Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues

Protein Coexpression Using FMDV 2A: Effect of “Linker” Residues

Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications

Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

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