Abstract
BackgroundHuman artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction. Application of Measles Virus (MV) fusogenic proteins to MMCT instead of polyethylene glycol (PEG) has partly solved this drawback, whereas the tropism of MV fusogenic proteins is restricted to human CD46- or SLAM-positive cells.ResultsHere, we show that retargeting of microcell fusion by adding anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of the MV-H protein improves the efficiency of MV-MMCT to human fibroblasts which originally barely express both native MV receptors, and are therefore resistant to MV-MMCT. Efficacy of chimeric fusogenic proteins was evaluated by the evidence that the HAC, tagged with a drug-resistant gene and an EGFP gene, was transferred from CHO donor cells into human fibroblasts. Furthermore, it was demonstrated that no perturbation of either the HAC status or the functions of transgenes was observed on account of retargeted MV-MMCT when another HAC carrying four reprogramming factors (iHAC) was transferred into human fibroblasts.ConclusionsRetargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors. Therefore, this technology could facilitate the systematic cell engineering by HACs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0142-z) contains supplementary material, which is available to authorized users.
Highlights
Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells
Construction and validation of Haals-αTfR To explore applicability of Measles Virus (MV)-microcell-mediated chromosome transfer (MMCT) to human fibroblasts, transferrin receptor (TfR) was selected as the target receptor in giving a new directivity for MV-H protein, because TfR is known to be ubiquitously expressed in all tissues [31]
Anti-TfR single chain antibody (scFvs) were fused to the C terminus of a quadruple mutated H protein (Haals: Y481A, R533A, S548L and F549S), which lacked the ability to bind both CD46 and SLAM [27,33], to validate the effect of anti-TfR scFvs on cell fusion more precisely (Figure 1B)
Summary
Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. It was demonstrated that higher efficiency of microcell fusion was achieved in some human cells by means of microcells which expressed MV-derived fusion machinery, both the hemagglutinin (H) protein and fusion (F) protein, as compared to PEG-induced fusion. Considering that the complex of the chimeric H protein and the F protein can induce whole cell fusion, we were keen to study whether the chimeric H protein was capable of mediating microcell fusion
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