학술논문
New [alpha]- and SIN [gamma]-retrovectors for safe transduction and specific transgene expression in pancreatic [beta] cell lines
Document Type
Report
Author
Source
BMC Biotechnology. June 17, 2019, Vol. 19 Issue 1
Subject
Language
English
ISSN
1472-6750
Abstract
Author(s): Olivier Albagli[sup.1] , Alicia Maugein[sup.1] , Lukas Huijbregts[sup.1] , Delphine Bredel[sup.1,3] , Géraldine Carlier[sup.1] , Patrick Martin[sup.2] and Raphaël Scharfmann[sup.1] Background Transferring DNA sequences through viral vectors represents an [...]
Background Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing [beta] cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian [alpha]- or mouse [gamma]-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into [beta] cells. Results We here describe two novel [alpha]- or SIN (Self-Inactivating) [gamma]-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN [gamma]-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the [alpha]-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian [beta] cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in [beta] over non-[beta] cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human [beta] cells. Finally, both [alpha]- and SIN [gamma]-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human [beta] cell lines. Conclusion Our novel [alpha]- and SIN [gamma]-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian [beta] cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of [beta] cell function and monitor changes in their differentiation status. Keywords: [alpha]-Retrovector, SIN [gamma]-retrovector, Pancreatic [beta] cell lines, EndoC-[beta] H2 cells, Rat insulin promoter, BXV1 xenotropic retrovirus, TVA gesicles
Background Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing [beta] cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian [alpha]- or mouse [gamma]-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into [beta] cells. Results We here describe two novel [alpha]- or SIN (Self-Inactivating) [gamma]-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN [gamma]-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the [alpha]-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian [beta] cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in [beta] over non-[beta] cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human [beta] cells. Finally, both [alpha]- and SIN [gamma]-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human [beta] cell lines. Conclusion Our novel [alpha]- and SIN [gamma]-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian [beta] cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of [beta] cell function and monitor changes in their differentiation status. Keywords: [alpha]-Retrovector, SIN [gamma]-retrovector, Pancreatic [beta] cell lines, EndoC-[beta] H2 cells, Rat insulin promoter, BXV1 xenotropic retrovirus, TVA gesicles