Cargando…
Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells
Hemophilia B occurs due to a deficiency in human blood coagulation factor IX (hFIX). Currently, no effective treatment for hemophilia B has been identified, and gene therapy has been considered the most appropriate treatment. Mesenchymal stem cells (MSCs) have homing abilities and low immunogenicity...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
D.A. Spandidos
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367337/ https://www.ncbi.nlm.nih.gov/pubmed/28112377 http://dx.doi.org/10.3892/mmr.2017.6131 |
_version_ | 1782517754681622528 |
---|---|
author | Li, Shu-Jun Luo, Ying Zhang, Le-Meng Yang, Wei Zhang, Guo-Gang |
author_facet | Li, Shu-Jun Luo, Ying Zhang, Le-Meng Yang, Wei Zhang, Guo-Gang |
author_sort | Li, Shu-Jun |
collection | PubMed |
description | Hemophilia B occurs due to a deficiency in human blood coagulation factor IX (hFIX). Currently, no effective treatment for hemophilia B has been identified, and gene therapy has been considered the most appropriate treatment. Mesenchymal stem cells (MSCs) have homing abilities and low immunogenicity, and therefore they may be potential cell carriers for targeted drug delivery to lesional tissues. The present study constructed an adeno-associated virus integration site 1 (AAVS1)-targeted vector termed AAVS1-green fluorescent protein (GFP)-hFIX and a zinc finger nuclease (ZFN) expression vector. Nucleofection was used to co-transfect the targeting vector and the ZFN expression vector into human MSCs. The GFP-positive cells were selected using flow cytometry. Site-specific integration clones were obtained following the monoclonal culture, subsequent detections were performed using polymerase chain reaction and Southern blotting. Following the confirmation of stem cell traits of the site-specific integration MSCs, the in vivo and in vitro expression levels of hFIX were detected. The results demonstrated that the hFIX gene was successfully transfected into the AAVS1 locus in human MSCs. The clones with the site-specific integration retained stem cell traits of the MSCs. In addition, hFIX was effectively expressed in vivo and in vitro. No significant differences in expression levels were identified among the individual clones. In conclusion, the present study demonstrated that the exogenous gene hFIX was effectively expressed following site-specific targeting into the AAVS1 locus in MSCs; therefore, MSCs may be used as potential cell carriers for gene therapy of hemophilia B. |
format | Online Article Text |
id | pubmed-5367337 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | D.A. Spandidos |
record_format | MEDLINE/PubMed |
spelling | pubmed-53673372017-04-13 Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells Li, Shu-Jun Luo, Ying Zhang, Le-Meng Yang, Wei Zhang, Guo-Gang Mol Med Rep Articles Hemophilia B occurs due to a deficiency in human blood coagulation factor IX (hFIX). Currently, no effective treatment for hemophilia B has been identified, and gene therapy has been considered the most appropriate treatment. Mesenchymal stem cells (MSCs) have homing abilities and low immunogenicity, and therefore they may be potential cell carriers for targeted drug delivery to lesional tissues. The present study constructed an adeno-associated virus integration site 1 (AAVS1)-targeted vector termed AAVS1-green fluorescent protein (GFP)-hFIX and a zinc finger nuclease (ZFN) expression vector. Nucleofection was used to co-transfect the targeting vector and the ZFN expression vector into human MSCs. The GFP-positive cells were selected using flow cytometry. Site-specific integration clones were obtained following the monoclonal culture, subsequent detections were performed using polymerase chain reaction and Southern blotting. Following the confirmation of stem cell traits of the site-specific integration MSCs, the in vivo and in vitro expression levels of hFIX were detected. The results demonstrated that the hFIX gene was successfully transfected into the AAVS1 locus in human MSCs. The clones with the site-specific integration retained stem cell traits of the MSCs. In addition, hFIX was effectively expressed in vivo and in vitro. No significant differences in expression levels were identified among the individual clones. In conclusion, the present study demonstrated that the exogenous gene hFIX was effectively expressed following site-specific targeting into the AAVS1 locus in MSCs; therefore, MSCs may be used as potential cell carriers for gene therapy of hemophilia B. D.A. Spandidos 2017-03 2017-01-19 /pmc/articles/PMC5367337/ /pubmed/28112377 http://dx.doi.org/10.3892/mmr.2017.6131 Text en Copyright: © Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
spellingShingle | Articles Li, Shu-Jun Luo, Ying Zhang, Le-Meng Yang, Wei Zhang, Guo-Gang Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells |
title | Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells |
title_full | Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells |
title_fullStr | Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells |
title_full_unstemmed | Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells |
title_short | Targeted introduction and effective expression of hFIX at the AAVS1 locus in mesenchymal stem cells |
title_sort | targeted introduction and effective expression of hfix at the aavs1 locus in mesenchymal stem cells |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367337/ https://www.ncbi.nlm.nih.gov/pubmed/28112377 http://dx.doi.org/10.3892/mmr.2017.6131 |
work_keys_str_mv | AT lishujun targetedintroductionandeffectiveexpressionofhfixattheaavs1locusinmesenchymalstemcells AT luoying targetedintroductionandeffectiveexpressionofhfixattheaavs1locusinmesenchymalstemcells AT zhanglemeng targetedintroductionandeffectiveexpressionofhfixattheaavs1locusinmesenchymalstemcells AT yangwei targetedintroductionandeffectiveexpressionofhfixattheaavs1locusinmesenchymalstemcells AT zhangguogang targetedintroductionandeffectiveexpressionofhfixattheaavs1locusinmesenchymalstemcells |