Stable lentiviral gene expression driven by constitutive promoters in mouse pluripotent stem cells
Suleiman
Y.
Alhaji1, 2, 3*,
Omar
Habib1, 2,
Shariza
Nordin1, 2,
Lai
M.
I1, 4,
Ngai
S.
Ching5 and
Syahril
Abdullah1, 2
-
1
Universiti Putra Malaysia, Genetics & Regenerative Medicine Research Centre, Faculty of Medicine & Health Sciences, Malaysia
-
2
Universiti Putra Malaysia, Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences , Malaysia
-
3
Abubakar Tafawa Balewa University Bauchi, Department of Human Anatomy, College of Medicine & Health Sciences, Nigeria
-
4
Universiti Putra Malaysia, Department of Pathology, Faculty of Medicine and Health Sciences, Malaysia
-
5
The University of Nottingham Malaysia, School of Biosciences, Faculty of Science, Malaysia
Lentiviral gene delivery system has proven to present significant transgene expression in mammalian somatic cells. However, the duration of the transgene expression is short-lived, which has been recognized as a major challenge in the realization of effective somatic cell gene therapy application. Here we provide a data demonstrating that lentiviral gene delivery in pluripotent stem cells, which has minimal DNA methylation profile, showed extended duration of transgene expression.
Constitutive promoters have been reported to efficiently drive transgene expression in mammalian somatic cells (Hong, Hwang et al. 2007, Norrman, Fischer et al. 2010). However, the duration of the transgene expression is short-lived, even in the integrative lentiviral gene delivery context. This has been recognized as a major challenge in the realization of effective somatic cell gene therapy application (Bestor 2000). Lentiviral gene delivery in pluripotent stem cells, which has minimal DNA methylation profile (Guo, Zhu et al. 2014), may show extended duration of transgene expression. We assessed the duration of transgene expression in mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells derived from the tail-tip fibroblast of C57BL/6 mice. Both cells were transduced with Lentivirus (LV) carrying Emerald Green fluorescent protein (EmGFP) driven by either Human elongation factor alpha (EF1) or Cytomegalovirus (CMV) promoter, since promoter activity can greatly vary between cell types as well as the loci into which the transgene is being expressed. LV/EF1α and LV/CMV transduced iPS cells exhibited significant GFP expression ( 80% expressing cells) with persistent level of mean fluorescent intensity MFI for 30 days. Both LV/EF1α and LV/CMV presented significant GFP expression (> 50%) in mouse ES cells for up to 30 days but surprisingly, LV/EF1α showed significantly higher MFI when compared to LV/CMV. Pluripotency analysis indicated that the cells retained their pluripotency potential throughout the study period.
In this study, the cells were transduced with optimal multiplicity of infection (MOI) of lentivirus. Primary tail-tip fibroblast cells from C57BL/6 mouse were used as a somatic cell control. The cells were sorted from non-GFP expressing cells two days post transduction using FACSAria III. Time point analysis was started two days post sorting in order to avoid detection of transgene expression from nonintegrated lentivirus. Pluripotency markers analysis by immunocytochemistry was performed. Functional pluripotency analysis by embroid body formation was conducted.
The results showed that iPSCs present significant GFP expression (80% expressing cells) with persistent level of MFI for up to 30 days and beyond. Surprisingly, ES cells presented lower percentage of GFP expression, with LV/EF1α showing significantly higher MFI when compared to LV/CMV. Functional pluripotency analysis indicated that the cells were able to form good quality EBs. The cells were also found to expressed OCT4 and SOX2 protein, indicating strong markers for pluripotency.
In conclusion, the research findings might provide an insight for improvement of gene delivery and regenerative medicine using pluripotent stem cells, especially iPSCs, for persistent correction of genetic related disorders.
Acknowledgements
1. Commonwealth Scholarship and Fellowship Plan (CSFP) Ministry of Higher Education Malaysia
2. Ministry of Science and Technology and Innovation (MOSTI) Universiti Putra Malaysia. Project Number: 02-01-04-SF1838
Keywords:
Embryonic Stem Cells,
Gene Expression,
Lentivirus,
iPS cells,
gene delivery
Conference:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016.
Presentation Type:
Poster
Topic:
Gene Therapy, Reprogramming and Pluripotency
Citation:
Alhaji
SY,
Habib
O,
Nordin
S,
I
LM,
Ching
NS and
Abdullah
S
(2016). Stable lentiviral gene expression driven by constitutive promoters in mouse pluripotent stem cells.
Front. Bioeng. Biotechnol.
Conference Abstract:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting.
doi: 10.3389/conf.FBIOE.2016.02.00008
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Received:
08 Dec 2016;
Published Online:
19 Dec 2016.
*
Correspondence:
Mr. Suleiman Y Alhaji, Universiti Putra Malaysia, Genetics & Regenerative Medicine Research Centre, Faculty of Medicine & Health Sciences, Serdang, Selangor, 43400, Malaysia, aysuleiman2002@yahoo.com