10 research outputs found
Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma
Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety.To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection.Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications
Treatment group and number of days from initiation of C-MYC overexpression until sacrifice (survival time) for each case of metastasis.
<p>Treatment group and number of days from initiation of C-MYC overexpression until sacrifice (survival time) for each case of metastasis.</p
Experimental design of tumorigenesis assay.
<p>(<b>a</b>) Transgenic mice were taken off of doxycycline drinking water at 7–8 weeks of age to induce expression of the human <i>C-MYC</i> transgene specifically in the liver from the LAP promoter, except for one control group (MYC off). Exactly one week after C-MYC induction, all groups except one control group (MYC on, no injection) were given hydrodynamic injections of phosphate-buffered saline alone or DNA plasmids diluted in phosphate-buffered saline. Mice were monitored weekly, imaged every two or three weeks, and sacrificed when tumors were detectable by gross distention of the abdomen as pictured. Inset shows the dissected liver and tumors from the pictured mouse, which was representative of all mice in all groups. (<b>b</b>) The plasmids given by hydrodynamic injection and their features.</p
Luciferase expression and the relationship between initial expression values and long-term survival.
<p>(<b>a</b>) <i>TRE-MYC/LAP-tTA</i> transgenic mice given pCS/pLiLucB (purple diamonds), pCSmI/pLiLucB (cyan squares), or pCSI/pLiLucB (blue triangles) by hydrodynamic injection were imaged at Day 1, Week 2, 4, 6, 8, and 10. The normalized luciferase levels were obtained by dividing the average luciferase expression in reflective light units (RLU) at each time point by the average level at day 1 for that group. Propagation of errors was used to determine the standard error at each time point given the division calculation (error bars). By student's t-test of the values at day 70, the pCSI/pLiLucB group and pCS/pLiLucB group had a p-value of 0.067, while the pCSI/pLiLucB group and pCS/pLiLucB group were significantly higher than the pCSmI/pLiLucB group (p = 0.014 and p = 0.000031, respectively). (<b>b</b>) Each mouse is represented by one point on the scatterplot, using the day 1 luciferase value in reflective light units (RLU) as the x-coordinate and the days of survival as the y-coordinate. The symbols and colors are identical to those used in <b>a</b>. The linear line-of-best-fit was calculated by GraphPad Prism and is plotted for each group (R-squared values of pCS/pLiLucB, 0.1065; pCSmI/pLiLucB, 0.3839; pCSI/pLiLucB, 0.0033). No R-squared values exceeded 0.95, which would have indicated that there was a trend relating transfection efficiency and survival.</p
Luciferase activity and PCR analysis of tumors from mice in the pCSI/pLiLucB group provide no evidence of φC31 integrase activity.
<p>(<b>a</b>) Protein extracts were prepared and the luciferase activity was measured in absolute counts per second (CPS). Controls included HeLa cells given FuGene 6 alone [HeLa (-)] or the CMV-luciferase plasmid pNBL2 via FuGene 6 [HeLa (+)], the normal-appearing part of the tumor-ridden liver taken from either a saline-injected mouse [Liver (-)] and pCSI/pLiLucB-injected mouse [Liver (+)]. Eight tumor samples (Tumor 1 through 8) and one metastasis (Met 1) that were obtained from four animals were also analyzed. The error bars give standard error of the mean for four replicates of each sample. (<b>b</b>) PCR analysis to detect the pLiLucB plasmid by amplification of the luciferase transgene. Plasmid DNA (20 ng pLiLucB) and no DNA controls show specific amplification of luciferase only in the reaction containing plasmid. One mouse each from the saline-only and pCSmI/pLiLucB groups was analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues (none found). Three mice in the pCSI/pLiLucB group were analyzed for transgene presence in normal-appearing (N) and tumor (T) tissues. Luciferase could be detected in 2/3 normal-appearing liver samples and none of the tumors. (<b>c</b>) PCR analysis for integration at the mpsL1 pseudo <i>attP</i> site was done on 18 tumors (lanes 5 through 22) and one metastasis (lane 23) taken from nine mice given pCSI/pLiLucB by hydrodynamic injection. Controls included no DNA (1<sup>st</sup> round, lane 1 and 2<sup>nd</sup> round, lane 25), and a DNeasy performed on no tissue (lane 2) to show no contamination from the DNA isolation procedure. Normal-appearing liver from a mouse in the pCSI/pLiLucB group (lane 3) served as the positive control. DNA isolated from a tumor in the saline-only group served as the negative control (lane 4). PCR for the <i>GAPDH</i> gene showed that sufficient DNA was added to all reactions. Seven tumors and one metastasis were subjected to the analysis in <i>both </i><b>a</b> and <b>c</b>. Six tumors were analyzed by all assays (<b>a</b>, <b>b</b> and <b>c</b>).</p
Survival curves suggest that hydrodynamic injection may contribute to C-MYC-induced tumor formation in the mouse liver.
<p>(<b>a</b>) Key showing the number of animals (n), median survival time in days (Median), and statistical results for each group. The significance as determined by the Gehan-Breslow-Wilcoxon test comparing each group to the MYC on, no injection (no inj?) or MYC on, saline injection (saline?) control groups is given. (<b>b</b>) A comparison of MYC off, no injection (yellow), MYC on, no injection (orange), and MYC on, saline injection (red) survival curves. (<b>c</b>) A comparison of MYC on, saline injection and MYC on, pCS/pLiLucB (purple) injection survival curves. (<b>d</b>) A comparison of MYC on, pCS/pLiLucB, MYC on, pCSmI/pLiLucB (cyan), and MYC on, pCSI/pLiLucB (blue) survival curves. (<b>e</b>) The survival curves of groups given pCSI with and without (green) donor plasmid. (<b>f</b>) A comparison of pCSI/pLiLucB to the control groups of no injection and saline-only injection. (<b>g</b>) The survival curves of all groups shown on the same plot. All plots and statistics were done using GraphPad Prism software.</p
Targeted Disruption of Hotair Leads to Homeotic Transformation and Gene Derepression
Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes
A Self-Limiting Switch Based on Translational Control Regulates the Transition from Proliferation to Differentiation in an Adult Stem Cell Lineage
Generation of pure GABAergic neurons by transcription factor programming
Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission