New α- and SIN γ-retrovectors for safe transduction and specific transgene expression in pancreatic β cell lines

Abstract

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 β cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into β cells. Results We here describe two novel α- or SIN (Self-Inactivating) γ-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 γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-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 β 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 β over non-β 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 β cells. Finally, both α- and SIN γ-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 β cell lines. Conclusion Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian β 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 β cell function and monitor changes in their differentiation status. Electronic supplementary material The online version of this article (10.1186/s12896-019-0531-9) contains supplementary material, which is available to authorized users.

Internal Promoters and Their Effects on the Transcription of Operon Genes for Epothilone Production in Myxococcus xanthus

“…In human spuma retrovirus (HSRV), an internal promoter was found in the HSRV operon, which was constitutively activated by the internal promoter and thus resulted in the accumulation of nonstructural proteins inside the host cell at the early stage of HSRV replication ( Löchelt et al, 1993 ; Xu et al, 2019 ). This kind of internal promoters has been reported for wide use in the construction of retroviral vectors, in which the internal promoter avoids the problems associated with the sequences in the intron that can interfere with splicing the message for the second gene ( Albagli et al, 2019 ). In the halophilic archaeon Haloferax volcanii , the rpl37R gene coding ribosomal L37.…”

The supply chain of human pancreatic β cell lines

“…The unexpected, residual RB pathway may restrict proliferation in one way, but it would also be expected to maintain RIP activity and thus SV40LT expression and proliferation. Indeed, RB knockdown in (SV40LT-expressing) EndoC-βH2 cells quells both their β identity and their RIP activity (117). As noted above, SV40LT expression is almost constant across the different human β cell lines, and even a small decrease (about 2-fold) in SV40LT expression reduces their proliferation and triggers an increase in P21 (CDKN1A), INSULIN, and IAPP expression (117).…”

“…A notable feature of EndoC-βH cell lines is the reversibility of their immortalized phenotype. Upon excision of both hTERT and SV40LT (70,71,116), or knockdown of SV40LT mRNA (21,117), immortalized human β cells exit the cell cycle and deepen β cell-specific traits. For example, they upregulate the expression of IAPP (encoding islet amyloid polypeptide), INSULIN, SLC2A2 (encoding the glucose transporter GLUT2), and the ABCC8 and KCNJ11 subunits of the ATP-sensitive potassium channel, which are targets of the sulfonylurea drugs, and they downregulate disallowed genes such as LDH-A and MCT-1 (70).…”

“…Indeed, RB knockdown in (SV40LT-expressing) EndoC-βH2 cells quells both their β identity and their RIP activity (117). As noted above, SV40LT expression is almost constant across the different human β cell lines, and even a small decrease (about 2-fold) in SV40LT expression reduces their proliferation and triggers an increase in P21 (CDKN1A), INSULIN, and IAPP expression (117). Thus, human β cell lines need SV40LT expression between a lower and upper threshold to ensure proliferation on one hand and conservation of the β cell phenotype and viability (with residual RB activity) on the other hand.…”

Guidelines for the antidiabetic assay for bioactive substances in cell model

“…Taken this under consideration, F I G U R E 3 Coculture of cells for antidiabetic assay NAKT-15 and BRIN-BD11, are currently functioning and stable for investigation of antidiabetic effect of bioactive compounds. The former was successfully established by transfecting human islet β cells with recombinant retroviral vector containing SV40 Tag and hTERT cDNAs (Albagli et al, 2019). However, there is a large demand for islet cells, and the operation is complicated, time-consuming and costly.…”