Figure 1.
Structure and modus operandi of recombinase-responsive lentivirus vector-based gene switch modules with the conventional (A) or the episomal design (B).
SIN LTR, SIN hybrid long terminal repeat composed of HIV-1 and RSV sequences; ψ, HIV-1 packaging signal; RTS, recombinase target site; pA, polyadenylation signal; dashed magenta lines, chromosomal DNA; magenta and green broken arrows, endogenous and exogenous promoter elements, respectively. See the main text section “Experimental rationale and design” for a detailed explanation of the figure.
Figure 2.
Diagram of the lentivirus vector shuttle plasmids pLV.pA+.GS.DsRed and pLV.pA+.GS.Luc.
Grey box with broken arrow, 5′ hybrid long terminal repeat (LTR) containing Rous sarcoma virus and HIV-1 sequences; Grey box without broken arrow, 3′ SIN LTR; ψ, HIV-1 packaging signal; Cyan arrowheads, FLP recognition target (FRT) sites; orange box with broken arrow, human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) gene promoter; Large and small green boxes, rabbit β-globin and murine metallothionine gene pAs (rBGpA and mMTpA, respectively); red box, DsRed.T4-N1 ORF from Discosoma sp. (DsRed); yellow box, luciferase ORF from Photinus pyralis (PpLuc). All genetic elements are drawn to scale.
Figure 3.
Functional testing of the lentivirus vectors LV.GS.DsRed and LV.GS.Luc.
(A) Direct fluorescence microscopy of human myoblasts not exposed (mock) or exposed to 3 different doses of LV.GS.DsRed (i.e. 3, 9 and 15 TU/cell) in the absence (no FLPe) or in the presence of FLPe (LV.FLPe or HD.FLPe). (B) Flow cytometric analysis of human myoblasts transduced with 3 different dosages of LV.GS.DsRed (i.e. 3, 9 and 15 TU/cell) and subsequently exposed to increasing amounts of FLPe-encoding viral vectors (i.e. LV.FLPe [open bars] and HD.FLPe [solid bars]) or not. Experimental conditions not tested are marked by an asterisk (*). (C) Dot plot representation of DsRed.T4-N1 expression in human myoblasts stably transduced with LV.DsRed (myoblastsGS.DsRed) in the absence (-FLPe) or presence (+FLPe) of HD.FLPe. (D and E) Luminometric analysis of luciferase activity in lysates derived from hMSCs or myoblasts mock-treated (D and E; hMSCsmock and myoblastsmock, respectively) and from hMSCs or myoblasts LV.GS.Luc-transduced (D and E; hMSCsGS.Luc and myoblastsGS.Luc, respectively) that were or were not exposed to different amounts of HD.FLPe particles. Graph bars shows mean ± standard error of the mean (n = 3). RLU, relative light units.
Figure 4.
Fusion-dependent reporter gene activation in an ex vivo human skeletal muscle cell differentiation system.
(A) Establishment of FLPe-positive human myoblast cultures. Phase-contrast microcopy of human myoblasts initially incubated with 0, 30, 300 and 900 µl of cleared culture supernatant from LV.FLPe.PurR-producing 293T cells. Micrographs were acquired 7 days posttransduction and 5 days after the addition of puromycin to a final concentration of 1.0 µg/ml. (B) Luminometric analysis of cell lysates derived from co-cultures of human myoblasts stably transduced with LV.FLPe.PurR or with LV.GS.Luc (myoblastsFLPe and myoblastsGS.Luc, respectively). The two types of human myoblast populations were mixed at the indicated ratios and luciferase activity was measured before (B) and after (A) myogenic differentiation. Bars represent mean ± standard error of the mean (n = 3). RLU, relative light units.
Figure 5.
Time-dependent reporter gene activation in an ex vivo human skeletal muscle cell differentiation system.
(A) Phase contrast microscopy of co-cultures containing myoblastsFLPe and myoblastsGS.Luc at a 1∶1 ratio after incubation for 2, 3, 4 or 5 days in growth medium (upper panels) or in differentiation medium (lower panels). (B) Luminometric analysis of cell lysates prepared at 12-hour intervals from co-cultures initiated with 5×104 myoblastsFLPe and with 5×104 myoblastsGS.Luc (upper panels) or with 105 myoblastsFLPe and with 105 myoblastsGS.Luc (lower panels). The cells were either maintained in growth medium (solid bars) or in differentiation medium (open bars). The relative light units (RLU) are plotted on linear (left panels) and logarithmic (right panels) scales. Bars correspond to mean ± standard error of the mean (n = 3).
Figure 6.
Probing the sensitivity of the lentivirus vector-based conditional gene expression assay to detect myoblast fusion.
(A) Phase contrast microscopic images of cultures started with a total number of 2×105 myoblasts. The pictures were taken after a 5-day incubation in differentiation medium. These cultures consisted exclusively of myoblastsGS.Luc (bar labeled 0∶100) or of myoblastsFLPe (bar labeled 100:0) or, contained mostly myoblastsGS.Luc (90–99.96%) spiked with different amounts of myoblastsFLPe (i.e. 0.04, 0.12, 0.37, 1.1, 3.3 and 10%). (B) Luciferase activity in cultures initiated with a total number of 2×105 myoblasts and exposed for 5 days to differentiation medium. The cultures were composed exclusively of myoblastsGS.Luc (grey bar) or of myoblastsFLPe (white bar) or contained myoblastsGS.Luc (90–99.96%) together with low proportions of myoblastsFLPe (i.e. 0.04, 0.12, 0.37, 1.1, 3.3 and 10%). Cumulative data are expressed as means ± standard error of the mean (n = 3). P values resulting from the comparison of relevant experimental groups were determined using an unpaired one-tailed Student's t-test. P values <0.05 were considered significant. RLU, relative light units.
Figure 7.
Pharmacological and genetic inhibition of the p38 MAPK pathway and its impact on human myotube formation in vitro.
(A) Luminometric analysis of cell lysates generated from co-cultures initiated with 105 myoblastFLPe and 105 myoblastGS.Luc cells and exposed for 3 days to differentiation medium (white bar), to differentiation medium supplemented with 0.1, 0.5 and 2.5 µM SB 203580 (black bars) or to differentiation medium containing a final concentration of vehicle equivalent to that applied to co-cultures incubated with 2.5 µM SB 203580 (grey bar). Cumulative data are presented as means ± standard deviations (n = 3). RLU, relative light units. (B) Western blot analysis of p38α levels in protein lysates of parental myoblasts and myoblastsGS.Luc (mock) and of myoblasts and myoblastsGS.Luc stably transduced with shRNA modules designed to down-regulate expression of eGFP (sh.eGFP), hif1α (sh.hif1α) and human p38α (sh.p38α.35, sh.p38α.33, sh.p38α.36 and sh.p38α.34). The α- and β-tubulins served as loading control. (C) Diagram outlining the experimental set-up applied to investigate the impact of post-transcriptional down-regulation of p38α expression on human myocyte fusion (see text for details). (D) Quantification through chemiluminescence of myoblast fusion activity in co-cultures consisting of a 1∶1 mixture of FLPe- and GS.Luc-encoding myoblasts either not transduced (none) or stably transduced with shRNAs sh.p38α.33, sh.p38α.36 or sh.hif1α. Data were derived from a minimum of 3 and a maximum of 6 different experiments and are presented as means ± standard error of the mean. RLU, relative light units.