Functional testing of the lentivirus vectors LV.GS.DsRed and LV.GS.Luc.

<p>(<b>A</b>) 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>B</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 (*). (<b>C</b>) Dot plot representation of <i>DsRed.T4-N1</i> expression in human myoblasts stably transduced with LV.DsRed (myoblasts^GS.DsRed) in the absence (-FLPe) or presence (+FLPe) of HD.FLPe. (<b>D and E</b>) Luminometric analysis of luciferase activity in lysates derived from hMSCs or myoblasts mock-treated (<b>D and E</b>; hMSCs^mock and myoblasts^mock, respectively) and from hMSCs or myoblasts LV.GS.Luc-transduced (<b>D and E</b>; hMSCs^GS.Luc and myoblasts^GS.Luc, respectively) that were or were not exposed to different amounts of HD.FLPe particles. Graph bars shows mean ± standard error of the mean (<i>n = </i>3). RLU, relative light units.</p

Structure and <i>modus operandi</i> of recombinase-responsive lentivirus vector-based gene switch modules with the conventional (A) or the episomal design (B).

<p>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.</p

Time-dependent reporter gene activation in an <i>ex vivo</i> human skeletal muscle cell differentiation system.

<p>(<b>A</b>) Phase contrast microscopy of co-cultures containing myoblasts^FLPe and myoblasts^GS.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>B</b>) Luminometric analysis of cell lysates prepared at 12-hour intervals from co-cultures initiated with 5×10⁴ myoblasts^FLPe and with 5×10⁴ myoblasts^GS.Luc (upper panels) or with 10⁵ myoblasts^FLPe and with 10⁵ myoblasts^GS.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 (<i>n = </i>3).</p

Diagram of the lentivirus vector shuttle plasmids pLV.pA+.GS.DsRed and pLV.pA+.GS.Luc.

<p>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 <i>glyceraldehyde-3-phosphate dehydrogenase</i> (hGAPDH) gene promoter; Large and small green boxes, rabbit <i>β-globin</i> and murine <i>metallothionine</i> gene pAs (rBGpA and mMTpA, respectively); red box, <i>DsRed.T4-N1</i> ORF from <i>Discosoma sp</i>. (DsRed); yellow box, <i>luciferase</i> ORF from <i>Photinus pyralis</i> (PpLuc). All genetic elements are drawn to scale.</p

Fusion-dependent reporter gene activation in an <i>ex vivo</i> human skeletal muscle cell differentiation system.

<p>(<b>A</b>) 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>B</b>) Luminometric analysis of cell lysates derived from co-cultures of human myoblasts stably transduced with LV.FLPe.PurR or with LV.GS.Luc (myoblasts^FLPe and myoblasts^GS.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 (<i>n = </i>3). RLU, relative light units.</p

Alignment of the nucleotide sequences immediately upstream of the <i>Luc</i> ORFs in pLV.GS.GpLuc.v1, pLV.GS.PpLuc and pLV.GS.GpLuc.v6.

<p>Blue box, 3′ end of the mMT1 pA; underlined sequences, out-of-frame ORFs preceding <i>Luc</i> ORFs; boxed TAA sequences, in-frame stop codons preceding <i>Luc</i> ORFs; red letters, in-frame ORFs preceding <i>Luc</i> ORFs; green letters, 5′ in-frame extension of the <i>GpLuc</i> ORF in pLV.GS.GpLuc.v1; black box, minimal FRT sequence; boxed ATG sequences, <i>Luc</i> initiation codons; light yellow box, 5′ end <i>GpLuc</i> ORF; dark yellow box, 5′ end <i>PpLuc</i> ORF.</p

Microscopic analysis of cell fusion kinetics in 1∶1 co-cultures of myoblasts-FLPe^NLS⁻ and myoblasts^GS.GLuc after maintenance for 72 h in growth medium and subsequent exposure to differentiation medium.

<p>At 24, 48, 72, 96 or 120 h after initiation of differentiation the cells were fixed and immunostained for skTnI (red fluorescence). The blue fluorescence corresponds to nuclei labeled with the karyophilic dye Hoechst 33342. The upper and lower row of pictures show phase-contrast images and fluoromicrographs, respectively. The first syncytia appeared at ±48 h after serum removal. The cell cultures displayed a time-dependent increase in frequency and size of myotubes/sacs until the cells started to detach from the surface of the culture plates. In parallel cultures of myoblasts kept in normal growth medium the cells remained firmly attached to their support and only few small syncytia were observed at late times after culture initiation (data no shown).</p

Analysis of GpLuc secretion in proliferating and differentiating 1∶1 co-cultures of myoblasts^GS.GLuc and either myoblasts-FLPe^NLS+ or myoblasts-FLPe^NLS⁻.

<p>(<b>A</b>): Luminometric analysis of culture medium of co-cultures of 50% myoblasts^GS.GLuc and 50% myoblasts-FLPe^NLS+ (+) or 50% myoblasts-FLPe^NLS− (−). At 72 h after cell seeding, the culture fluid was replaced by fresh culture medium with (growth conditions, no differentiation) or without (differentiation conditions) serum, which was left on the cells for the indicated 24-h time intervals. Bars represent mean ± standard error of the mean (n = 3). (<b>B</b>): Fold change in luciferase activity calculated on the basis of the data presented in (A). For each sampling time the average light production under growth conditions was the denominator and the mean of the RLUs produced under differentiation conditions was the numerator. RLUs, relative light units; NLS, nuclear localization signal.</p

Structure of the LV DNA in the LV shuttle plasmids.

<p>(<b>A</b>): pLV.hCMV-IE.FLPe^NLS+.IRES.PurR.hHBVPRE (<b>B</b>): pLV.hCMV-IE.FLPe^NLS−.IRES.PurR.hHBVPRE (<b>C</b>): pLV.hCMV-IE.IRES.PurR.hHBVPRE and (<b>D</b>): pLV.GS.GpLuc.v1. The start codons of the FLPe^NLS+ and FLPe^NLS− ORFs are shown in boldface. 5′ LTR, chimeric 5′ long terminal repeat containing the Rous sarcoma virus U3 region and the HIV1 R and U5 regions; Ψ, HIV1 packaging signal; RRE, HIV1 Rev-responsive element; cPPT, HIV1 central polypurine tract and termination site; hCMV-IE, human cytomegalovirus <i>immediate early</i> gene promoter; FLPe^NLS+, molecularly evolved flippase with simian virus 40 (SV40) nuclear localization signal (NLS; black bar); FLPe^NLS−, molecularly evolved flippase without NLS; EMCV IRES, encephalomyocarditis virus internal ribosomal entry site; PurR; <i>Streptomyces alboniger</i> puromycin N-acetyl transferase-coding sequence; hHBVPRE, human hepatitis B virus posttranscriptional regulatory element; black triangle/FRT, flippase recognition target sequence; hGAPDH, human <i>glyceraldehyde 3-phosphate dehydrogenase</i> gene promoter; rHBB2 pA, rabbit <i>β-hemoglobin</i> gene polyadenylation signal; GpLuc, <i>Gaussia princeps</i> luciferase-coding sequence; mMT1 pA, mouse <i>metallothionein 1</i> gene polyadenylation signal; 3′ LTR, 3′ HIV1 long terminal repeat containing a deletion in the U3 region to render the LV self-inactivating.</p

Improved design of the <i>GpLuc</i> gene switch cassette.

<p>(<b>A–C</b>): Detailed structure of the areas upstream of the <i>Luc</i> ORFs in pLV.GS.GpLuc.v1 (A), pLV.GS.GpLuc.v6 (B) and pLV.GS.PpLuc (C) starting at the HIV1 3′ LTR. U5, HIV1 LTR unique 5′ region; R, HIV1 LTR repeat region; ΔU3, enhancer- and promoterless HIV1 LTR unique 3′ region; blue arrow, mouse <i>metallothionein 1</i> gene polyadenylation signal (mMT1 pA); small black triangle, AATAAA motif in mMT1 pA; red diamonds, stop codons in frame with <i>Luc</i> ORFs; large black triangle, minimal FRT sequence; light yellow arrow, <i>GpLuc</i> ORF; green box, 5′ in-frame extension of the <i>GpLuc</i> ORF; white arrows, out-of-frame ORFs preceding <i>Luc</i> ORFs; red arrows, in-frame ORFs preceding <i>Luc</i> ORFs; dark yellow arrow, <i>PpLuc</i> ORF.</p